Compositions and methods for prolonging survival of platelets

ABSTRACT

The present invention provides modified platelets having a reduced platelet clearance and methods for reducing platelet clearance. Also provided are compositions for the preservation of platelets. The invention also provides methods for making a pharmaceutical composition containing the modified platelets and for administering the pharmaceutical composition to a mammal to mediate hemostasis.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 60/424,807, entitled “Compositions andMethods for Prolonging Survival of Platelets,” filed on Nov. 8, 2002,which is herein incorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH

This invention was funded in part under National Institute of HealthGrants Nos. HL19429 and HL56949. The government may retain certainrights in the invention.

FIELD OF THE INVENTION

The inventions relate to compositions and methods for reducing theclearance of platelets and prolonging the survival of platelets.

BACKGROUND OF THE INVENTION

Platelets are anucleate bone marrow-derived blood cells that protectinjured mammals from blood loss by adhering to sites of vascular injuryand by promoting the formation of plasma fibrin clots. Humans depletedof circulating platelets by bone marrow failure suffer from lifethreatening spontaneous bleeding, and less severe deficiencies ofplatelets contribute to bleeding complications following trauma orsurgery.

A reduction in the number of circulating platelets to below ˜70,000 perμL reportedly results in a prolongation of a standardized cutaneousbleeding time test, and the bleeding interval prolongs, extrapolating tonear infinity as the platelet count falls to zero. Patients withplatelet counts of less than 20,000 per μL are thought to be highlysusceptible to spontaneous hemorrhage from mucosal surfaces, especiallywhen the thrombocytopenia is caused by bone marrow failure and when theaffected patients are ravaged with sepsis or other insults. The plateletdeficiencies associated with bone marrow disorders such as aplasticanemia, acute and chronic leukemias, metastatic cancer but especiallyresulting from cancer treatment with ionizing radiation and chemotherapyrepresent a major public health problem. Thrombocytopenia associatedwith major surgery, injury and sepsis also eventuates in administrationof significant numbers of platelet transfusions.

A major advance in medical care half a century ago was the developmentof platelet transfusions to correct such platelet deficiencies, and over9 million platelet transfusions took place in the United States alone in1999 (Jacobs et al., 2001). Platelets, however, unlike all othertransplantable tissues, do not tolerate refrigeration, because theydisappear rapidly from the circulation of recipients if subjected toeven very short periods of chilling, and the cooling effect thatshortens platelet survival is irreversible (Becker et al., 1973; Bergeret al., 1998).

The resulting need to keep these cells at room temperature prior totransfusion has imposed a unique set of costly and complex logisticalrequirements for platelet storage. Because platelets are activelymetabolic at room temperature, they require constant agitation in porouscontainers to allow for release of evolved CO₂ to prevent the toxicconsequences of metabolic acidosis. Room temperature storage conditionsresult in macromolecular degradation and reduced hemostatic functions ofplatelets, a set of defects known as “the storage lesion” (Chemoff andSnyder, 1992). But the major problem with room-temperature storage,leading to its short (5-day) limitation, is the higher risk of bacterialinfection. Bacterial contamination of blood components is currently themost frequent infectious complication of blood component use, exceedingby far that of viral agents (Engelfriet et al., 2000). In the USA,3000-4500 cases yearly of bacterial sepsis occur because of bacteriallycontaminated blood components (Yomtovian et al., 1993).

The mechanism underlying the unique irreversible cold intolerance ofplatelets has been a mystery as has its physiological significance.Circulating platelets are smooth-surfaced discs that convert to complexshapes as they react to vascular injury. Over 40 years ago investigatorsnoted that discoid platelets also change shape at refrigerationtemperatures (Zucker and Borrelli, 1954). Subsequent evidence that adiscoid shape was the best predictor of viability for platelets storedat room temperature (Schlichter and Harker, 1976) led to the conclusionthat the cold-induced shape change per se was responsible for the rapidclearance of chilled platelets. Presumably irregularly-shaped plateletsdeformed by cooling became entrapped in the microcirculation.

Based on our studies linking signaling to the mechanisms leading toplatelet shape changes induced by ligands (Hartwig et al., 1995), wepredicted that chilling, by inhibiting calcium extrusion, could elevatecalcium levels to a degree consistent with the activation of the proteingelsolin, which severs actin filaments and caps barbed ends of actinfilaments. We also reasoned that a membrane lipid phase transition atlow temperatures would cluster phosphoinositides. Phosphoinositideclustering uncaps actin filament barbed ends (Janmey and Stossel, 1989)to create nucleation sites for filament elongation. We producedexperimental evidence for both mechanisms, documenting gelsolinactivation, actin filament barbed end uncapping, and actin assembly incooled platelets (Hoffmeister et al., 2001; Winokur and Hartwig, 1995).Others have reported spectroscopic changes in chilled plateletsconsistent with a membrane phase transition (Tablin et al., 1996). Thisinformation suggested a method for preserving the discoid shape ofchilled platelets, using a cell-permeable calcium chelator to inhibitthe calcium rise and cytochalasin B to prevent barbed end actinassembly. Although addition of these agents retained platelets in adiscoid shape at 4° C. (Winokur and Hartwig, 1995), such platelets alsoclear rapidly from the circulation, as we report here. Therefore, theproblem of the rapid clearance of chilled platelets remains, and methodsof increasing circulation time as well as storage time for platelets areneeded.

SUMMARY OF THE INVENTION

The present invention provides modified platelets having a reducedplatelet clearance and methods for reducing platelet clearance. Alsoprovided are compositions for the preservation of platelets. Theinvention also provides methods for making a pharmaceutical compositioncontaining the modified platelets and for administering thepharmaceutical composition to a mammal to mediate hemostasis.

It has now been discovered that cooling of human platelets causesclustering of the von Willebrand factor (vWf) receptor complex α subunit(GP1bα) complexes on the platelet surface. The clustering of GP1bαcomplexes on the platelet surface elicits recognition by Macrophagecomplement type three receptors (αMβ2, CR3) in vitro and in vivo. CR3receptors recognize N-linked sugars with terminal βG1cNAc on the ofGP1bα complexes and phagocytose the platelets, clearing them from thecirculation and resulting in a concomitant loss of hemostatic function.

Applicants have discovered that treatment of platelets with certainsugar molecules, which is believed to lead to glycation of the exposedβG1cNAc residues on GP1bα reduced platelet clearance, blocked plateletphagocytosis, increased platelet circulation time, and increasedplatelet storage time.

According to one aspect of the invention, methods for increasing thecirculation time of a population of platelets is provided. The methodcomprises contacting an isolated population of platelets with at leastone glycan modifying agent in an amount effective to reduce theclearance of the population of platelets. In some embodiments, theglycan modifying agent is selected from the group consistingUDP-galactose and UDP-galactose precursors. In some preferredembodiments, the glycan modifying agent is UDP-galactose.

In some embodiments, the method further comprises adding an enzyme thatcatalyzes the modification of a glycan moiety. One example of an enzymethat catalyzes the modification of a glycan moiety is galactosyltransferase.

In one of the preferred embodiment, the glycan modifying agent isUDP-galactose and the enzyme that catalyzes the modification of theglycan moiety is galactosyl transferase.

In some embodiments, the method for increasing the circulation time of apopulation of platelets further comprises chilling the population ofplatelets prior to, concurrently with, or after contacting the plateletswith the at least one glycan modifying agent.

In some embodiments, the population of platelets retains substantiallynormal hemostatic activity.

In some embodiments, the step of contacting the population of plateletswith at least one glycan modifying agent is performed in a platelet bag.

In some embodiments, the circulation time is increased by at least about10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 100%, 150%, 200%, or more.

According to another aspect of the invention, a method for increasingthe storage time of platelets is provided. The method comprisescontacting an isolated population of platelets with an amount of atleast one glycan modifying agent effective to reduce the clearance ofthe population of platelets, and storing the population of platelets.

In some embodiments, the glycan modifying agent is selected from thegroup consisting of: UDP-galactose and UDP-galactose precursors. In somepreferred embodiments, the glycan modifying agent is UDP-galactose.

In some embodiments, the method further comprises adding an enzyme thatcatalyzes the addition of the glycan modifying agent to a glycan on thesurface of the platelets. In one of the preferred embodiments, theglycan modifying agent is UDP-galactose and the enzyme that catalyzesthe addition of the glycan modifying agent to a glycan on the surface ofthe platelets is galactosyl transferase.

In some embodiments, the method further comprises chilling thepopulation of platelets prior to, concurrently with, or after contactingthe platelets with the at least one glycan modifying agent.

In some embodiments, the population of platelets retains substantiallynormal hemostatic activity.

The step of contacting the population of platelets with at least oneglycan modifying agent is performed in a platelet bag.

The platelets are stored chilled for at least about 3 days, at leastabout 5 days, at least about 7 days, at least about 10 days, at leastabout 14 days, at least about 21 days, or at least about 28 days.

According to another aspect of the invention, a modified platelet isprovided. The modified platelet comprises a plurality of modified glycanmolecules on the surface of the platelet.

In some embodiments, the modified glycan molecules are moieties of GP1bαmolecules. The modified glycan molecules comprise at least one addedsugar molecule. The added sugar may be a natural sugar or may be anon-natural sugar.

Examples of added sugars include but are not limited to: UDP-galactoseand UDP-galactose precursors. In one of the preferred embodiments, theadded sugar is UDP-galactose.

In another aspect, the invention provides a platelet compositioncomprising a plurality of modified platelets. In some embodiments, theplatelet composition further comprises a storage medium. In someembodiments, the platelet composition further comprises apharmaceutically acceptable carrier.

According to yet another aspect of the invention, a method for making apharmaceutical composition for administration to a mammal is provided.The method comprises the steps of:

(a) contacting a population of platelets contained in apharmaceutically-acceptable carrier with at least one glycan modifyingagent to form a treated platelet preparation,

(b) storing the treated platelet preparation, and

(c) warming the treated platelet preparation.

In some embodiments, the step of warming the treated plateletpreparation is performed by warming the platelets to 37° C.

In some embodiments, the step of contacting a population of plateletscontained in a pharmaceutically-acceptable carrier with at least oneglycan modifying agent comprises contacting the platelets with at leastone glycan modifying agent in the presence of an enzyme that catalyzesthe modification of a glycan moiety. In some embodiments, the methodfurther comprises removing or neutralizing the enzyme in the plateletpreparation. Methods of removing or neutralizing the enzyme include, forexample, washing the platelet preparation.

Examples of glycan modifying agents are listed above. In one of thepreferred embodiments, the glycan modifying agent is UDP-galactose. Insome embodiments, the method further comprises adding an enzyme thatcatalyzes the addition of the glycan modifying agent to a glycan moiety.

In one of the preferred embodiments, the glycan modifying agent isUDP-galactose and the enzyme is galactosyl transferase.

In some embodiments, the population of platelets retain substantiallynormal hemostatic activity.

In certain embodiments, the step of contacting the population ofplatelets with at least one glycan modifying agent is performed in aplatelet bag.

In some embodiments, the platelet preparation is stored at a temperatureof less than about 15° C. In some other embodiments, the plateletpreparation is stored at room temperature.

According to yet another aspect of the invention, a method for mediatinghemostasis in a mammal is provided. The method comprises administering aplurality of modified platelets or a modified platelet composition tothe mammal.

According to still yet another aspect of the invention, a storagecomposition for preserving platelets is provided. The compositioncomprises at least one glycan modifying agent in an amount sufficient tomodify glycans to increase the storage time and/or the circulation timeof platelets added to the storage composition.

In some embodiments the composition further comprises an enzyme thatcatalyzes the modification of a glycan moiety.

In some embodiments, the composition is stored at a temperature of lessthan about 15° C. In some other embodiments, the composition is storedat room temperature.

According to another aspect of the invention, a container for collecting(and optionally processing) platelets is provided. The containercomprises at least one glycan modifying agent in an amount sufficient tomodify glycans of platelets added to the storage composition.

In some embodiments, the container further comprises an enzyme thatcatalyzes the modification of a glycan moiety with the glycan modifyingagent.

In some embodiments the container further comprises a plurality ofplatelets or plasma comprising a plurality of platelets.

In some embodiments, the glycan modifying agent is present at aconcentration higher than it is in naturally occurring platelets.

According to still yet another aspect of the invention, a device forcollecting and processing platelets is provided. The device comprises: acontainer for collecting platelets; at least one satellite container influid communication with said container; and at least one glycanmodifying agent in the satellite container.

In some embodiments, the glycan modifying agent in the satellitecontainer is present in sufficient amounts to preserve the platelets inthe container.

In some embodiments, the glycan modifying agent in the satellitecontainer is prevented from flowing into the container by a breakableseal.

These and other aspects of the invention, as well as various advantagesand utilities, will be more apparent in reference to the followingdetailed description of the invention. Each of the limitations of theinvention can encompass various embodiments of the invention. It istherefore, anticipated that each of the limitation involving any oneelement or combination of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows circulation time in mice of room temperature platelets andof platelets chilled and rewarmed in the presence or absence of EGTA-AMand Cytochalasin B. The curves depict the survival of5-chloromethylfluorescein diacetate (CMFDA) labeled, room temperature(RT) platelets, platelets chilled at ice-bath temperature (Cold) andrewarmed to room temperature before injection and chilled and rewarmedplatelets treated with EGTA-AM and cytochalasin B (Cold+CytoB/EGTA) topreserve their discoid shape. Each curve represents the mean±SD of 6mice. Identical clearance patterns were observed with ¹¹¹Indium-labeledplatelets.

FIG. 1B shows that chilled platelets aggregate normally in vitro.Washed, chilled-rewarmed (Cold) or room temperature (RT) wild typeplatelets were stimulated by the addition of the indicated agonists at37° C. and light transmission was recorded on a standard aggregometer.Aggregation responses of chilled platelets treated with EGTA-AM andcytochalasin B were identical to untreated chilled platelets.

FIG. 1C shows that cold induced clearance occurs predominantly in theliver of mice. The liver is the primary clearance organ of chilledplatelets, containing 60-90% of injected platelets. In contrast, RTplatelets are cleared more slowly in the spleen. ¹¹¹Indium labeledplatelets were injected into syngeneic mice and tissues were harvestedat 0.5, 1 and 24 hours. Data are expressed per gram of tissue. Each bardepicts the mean values of 4 animals analyzed±SD.

FIG. 1D shows that chilled platelets co-localize with hepatic sinusoidalmacrophages (Kupffer cells). This representative confocal-micrographshows the hepatic distribution of CMFDA-labeled, chilled-rewarmedplatelets (green) after 1 hour of transfusion, which preferentiallyaccumulate in periportal and midzonal fields of liver lobules. Kupffercells were visualized after injection of nile red-labeled spheres. Themerged micrograph that shows co-localization of chilled platelets andmacrophages in yellow. The lobule organization is indicated (CV: centralvein; PV: portal vein, bar: 100 μM).

FIG. 2 shows that chilled platelets circulate normally in CR3-deficientmice, but not in complement 3 (C3) or vWf deficient mice. CMFDA-labeledchilled-rewarmed (Cold) and room temperature (RT) wild type plateletswere transfused into six each of syngeneic wild type (WT), CR3-deficient(A), vWf-deficient (B) and C3-deficient (C) recipient mice and theirsurvival times determined. Chilled platelets circulate in CR3-deficientanimals with the same kinetics as room-temperature platelets, but arecleared rapidly from the circulation of C3- or vWf-deficient mice. Dataare mean±SD for 6 mice.

FIG. 3 shows that chilled platelets adhere tightly to CR3-expressingmouse macrophages in vivo. FIG. 3A—Chilled-rewarmed TRITC-labeledplatelets (left panel) adhere with a 3-4× higher frequency to liversinusoids than room temperature CMFDA-labeled platelets (right panel).The intravital fluorescence micrographs were obtained 30 min after theinfusion of the platelets. FIG. 3B—Chilled-rewarmed (Cold, open bars)and room temeperature platelets (RT, filled bars) adhere to sinusoidalregions with high macrophage density (midzonal) with similardistributions in wild type mice. FIG. 3C—Chilled-rewarmed plateletsadhere 3-4× more than room temperature platelets to macrophages in thewild type liver (open bars). In contrast, chilled-rewarmed or roomtemperature platelets have identical adherence to macrophages inCR3-deficient mice (filled bars). 9 experiments with wild type mice and4 experiments with CR3-deficient mice are shown (mean±SEM, *P<0.05:**P<0.01).

FIG. 4 shows that GPIbα mediates chilled platelet clearance, aggregatesin the cold, but binds activated vWf normally on chilled platelets. FIG.4A—CMFDA-labeled platelets enzymatically cleared of the GP1bαextracellular domain (left panel, inset, filled area) or controlplatelets were kept at room temperature (left panel) or chilled-rewarmed(right panel) infused into syngeneic wild type mice, and plateletsurvivals were determined. Each survival curve represents the meanvalues±SD for 6 mice. FIG. 4B—Chilled, or RT platelet rich plasma wastreated with (shaded area) or without (open area) botrocetin. vWf boundwas detected using FITC labeled anti-vWf antibody. FIG. 4C—The vWfreceptor redistributes from linear arrays (RT) into aggregates (Chilled)on the surface of chilled murine platelets. Fixed, chilled-rewarmed, orroom temperature platelets (RT) were incubated with monoclonal ratanti-mouse GPIbα antibodies followed by 10 nm colloidal gold particlescoated with goat anti-rat IgG. The bars are 100 nm. Inset: lowmagnification of platelets.

FIG. 5 shows GPIbα-CR3 interaction mediates phagocytosis of chilledhuman platelets in vitro. FIGS. 5A and 5B show a representative assayresult of THP-1 cells incubated with room temperature (RT) (FIG. 5A) orchilled-rewarmed (Cold) platelets (FIG. 5B). CM-Orange-labeled plateletsassociated with macrophages shift in orange fluorescence up the y axis.The mean percentage of the CM-Orange positive native macrophagesincubated with platelets kept at room temperature was normalized to 1.Chilling of platelets increases this shift from ˜4% to 20%. Theplatelets are predominantly ingested, because they do not dual labelwith the FITC-conjugated mAb to CD61. FIG. 5C Undifferentiated (openbars) THP-1 cells express ˜50% less CR3, and ingest half as manychilled-rewarmed platelets. Differentiation (filled bars) of CR3expression however, had no significant effect on the uptake of RTplatelets. Treatment of human platelets with the snake venommetalloprotease, mocarhagin (Moc), which removes the N-terminus of GP1bαfrom the surface of human platelets (inset; control: solid line,mocarhagin treated platelets: shaded area), reduced phagocytosis ofchilled platelets by ˜98%. Data shown are means±SD of 5 experiments.

FIG. 6 shows circulating, chilled platelets have hemostatic function inCR3 deficient mice. Normal in vivo function of room temperature (RT)platelets transfused into wild type mice (FIGS. 6A and 6B) and ofchilled (Cold) platelets transfused into CR-3 deficient mice (FIGS. 6Cand 6D), as determined by their equivalent presence in plateletaggregates emerging from the wound 24 hrs after infusion of autologousCMFDA labeled platelets. Peripheral blood (FIGS. 6A and 6C) and theblood emerging from the wound (shed blood, FIGS. 6B and 6D) wereanalyzed by whole blood flow cytometry. Platelets were identified byforward light scatter characteristics and binding of the PE-conjugatedanti-GPIbα mAb (pOp4). The infused platelets (dots) were identified bytheir CMFDA fluorescence and the non-infused platelets (contour lines)by their lack of CMFDA fluorescence. In the peripheral whole bloodsamples, analysis regions were plotted around the GPIbα-positiveparticles to include 95% of the population on the forward scatter axis(region 1) and the 5% of particles appearing above this forward lightscatter threshold were defined as aggregates (region 2). The percentagesrefer to the number of aggregates formed by CMFDA-positive platelets.This shown result is representative of 4 experiments. FIG. 6E shows exvivo function of CM-Orange, room temperature (RT) platelets transfusedinto wild type mice and CM-Orange, chilled-rewarmed (Cold) plateletstransfused into CR3 deficient mice, as determined by exposure ofP-selectin and fibrinogen binding following thrombin (1 U/ml) activationof blood drawn from the mice after 24 hours post infusion. CM-Orangelabeled platelets have a circulation half-life time comparable to thatof CMFDA labeled platelets (not shown). Transfused platelets wereidentified by their CM-Orange fluorescence (filled bars). Non-transfused(non-labeled) analyzed platelets are represented as open bars. Resultsare expressed as the percentage of cells present in the P-selectin andfibrinogen positive regions (region 2). Data are mean±SD for 4 mice.

FIG. 7 is a schematic depicting two platelet clearance pathways.Platelets traverse central and peripheral circulations, undergoingreversible priming at lower temperatures at the body surface. Repeatedpriming leads to irreversible GPIb-IX-V (vWfR) receptor complexreconfiguration and clearance by complement receptor type 3 (CR3)bearing hepatic macrophages. Platelets are also cleared after theyparticipate in microvascular coagulation.

FIG. 8 shows the effect of monosaccharides on phagocytosis of chilledplatelets.

FIG. 9 shows the dot plots of binding of WGA lectin to room temperatureplatelets or chilled platelets.

FIG. 10 shows the analysis of various FITC labeled lectins bound to roomtemperature or chilled platelets.

FIG. 11A shows the summary of FITC-WGA binding to the surface of roomtemperature or chilled platelets obtained by flow cytometry before andafter β-hexosaminidase treatment.

FIG. 11B shows that GPIbα removal from the platelet surface reducedFITC-WGA binding to chilled platelets.

FIG. 12 shows that galactose transfer onto platelet oligosaccharidesreduces chilled platelet (Cold) phagocytosis, but does not affect thephagocytosis of room temperature (RT) platelets.

FIG. 13 shows the survival of chilled, galactosylated murine plateletsrelative to untreated platelets.

FIG. 14 shows that platelets containing galactose transferases on theirsurface transfer galactose without the addition of external transferasesas judged by WGA binding (FIG. 14A) and in vitro phagocytosis resultsfor human platelets (FIG. 14B). FIG. 14C shows that of UDP-galactosewith or without Galactose transferase (GalT) on survival of murineplatelets. UDP-galactose with or without GalT was added to murineplatelets before chilling for 30 min at 37° C. The platelets werechilled for 2 hours in an ice bath and then transfused (10⁸platelets/mouse) into mice and their survival determined.

FIG. 15 shows the time course of ¹⁴C-labeled UDP-galactose incorporationinto human platelets.

FIG. 16 shows galactosylation of platelets in four platelet concentratesamples at different concentrations of UDP-galactose.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a population of modified platelets that haveenhanced circulation properties and that retain substantially normal invivo hemostatic activity. Hemostatic activity refers broadly to theability of a population of platelets to mediate bleeding cessation.Various assays are available for determining platelet hemostaticactivity (Bennett, J. S. and Shattil, S. J., 1990, “Platelet function,”Hematology, Williams, W. J., et al., Eds. McGraw Hill, pp 1233-12250).However, demonstration of “hemostasis” or “hemostatic activity”ultimately requires a demonstration that platelets infused into athrombocytopenic or thrombopathic (i.e., non-functional platelets)animal or human circulate and stop natural or experimentally-inducedbleeding.

Short of such a demonstration, laboratories use in vitro tests assurrogates for determining hemostatic activity. These tests, whichinclude assays of aggregation, secretion, platelet morphology andmetabolic changes, measure a wide variety of platelet functionalresponses to activation. It is generally accepted in the art that the invitro tests are reasonably indicative of hemostatic function in vivo.

Substantially normal hemostatic activity refers to an amount ofhemostatic activity that is about the same as the hemostatic activity ofa freshly isolated population of platelets.

The instant invention provides methods for increasing circulation timeof a population of platelets and increasing the storage time ofplatelets. Also provided are platelet compositions methods andcompositions for the preservation of platelets with preserved hemostaticactivity as well as methods for making a pharmaceutical compositioncontaining the preserved platelets and for administering thepharmaceutical composition to a mammal to mediate hemostasis.

In one aspect of the invention, the method for increasing circulationtime of an isolated population of platelets involves contacting anisolated population of platelets with at least one glycan modifyingagent in an amount effective to reduce the clearance of the populationof platelets. As used herein, a population of platelets refers to one ormore platelets. A population of platelets includes a plateletconentrate. The term “isolated” means separated from its nativeenvironment and present in sufficient quantity to permit itsidentification or use. As used herein with respect to a population ofplatelets, isolated means removed from blood circulation. Thecirculation time of a population of platelets is defined as the timewhen one-half of the platelets are no longer circulating. As usedherein, “clearance” means removal of platelets from blood circulation(such as by macrophage phagocytosis).

A glycan modifying agent refers to an agent that modifies terminalglycan residues recognized by macrophages, preferably on GP1bα, suchthat the macrophages no longer phagocytose the platelets. As usedherein, a “glycan” or “glycan residue” is a polysaccharide moiety onsurface of the platelet. A “terminal” glycan or glycan residue is theglycan at the terminus of a polysaccharide, which typically is attachedto polypeptides on the platelet surface.

In some embodiments, the glycan modifying agent is selected from thegroup consisting of uridine diphosphate galactose (UDP-galactose) andUDP-galactose precursors. In some preferred embodiments, the glycanmodifying agent is UDP-galactose. UDP-galactose is an intermediate ingalactose metabolism, formed by the enzymeUDP-glucose-α-D-galactose-1-phosphate uridylyltransferase whichcatalyzes the release of glucose-1-phosphate from UDP-glucose inexchange for galactose-1-phosphate to make UDP-galactose. Methods forsynthesis and production of UDP-galactose are well known in the art anddescribed in the literature (see for example, Liu et al, ChemBioChem 3,348-355, 2002; Heidlas et al, J. Org. Chem. 57, 152-157; Butler et al,Nat. Biotechnol. 8, 281-284, 2000; Koizumi et al, Carbohydr. Res. 316,179-183, 1999; Endo et al, Appl. Microbiol., Biotechnol. 53, 257-261,2000). UDP-galactose precursors are molecules, compounds, orintermediate compounds that may be converted (e.g., enzymatically orbiochemically) to UDP-galactose. One non-limiting example of aUDP-galactose precursor is UDP-glucose. In certain embodiments, anenzyme that converts a UDP-galactose precursor to UDP-galactose is addedto a reaction mixture (e.g. in a platelet container).

An effective amount of a glycan modifying agent means, that amount ofthe glycan modifying agent that increases circulation time and/orreduces the clearance of a population of platelets by modification of asufficient number of glycan residues on the surface of platelets.

Modification of platelets with UDP-galactose can be preformed asfollows. The population of platelets is incubated with differentconcentrations (1-1000 μM) of UDP-galactose for at least 1, 2, 5, 10,20, 40, 60, 120, 180, 240, or 300 min. at 22° C.-37° C. In someembodiments 0.1-500 mU/ml galactose transferase is added to thepopulation of platelets. Galactose transfer can be monitoredfunctionally using FITC-WGA (wheat germ agglutinin) binding. The goal ofthe glycan modification reaction is to reduce WGA binding to restingroom temperature WGA binding-levels. Galactose transfer can bequantified using ¹⁴C-UDP-galactose. Non-radioactive UDP-galactose ismixed with ¹⁴C-UDP-galactose to obtain appropriate galactose transfer.The transfer reaction is performed as described above. Platelets areextensively washed, and the incorporated radioactivity measured using aγ-counter. The measured cpm permits calculation of the incorporatedgalactose.

As used herein, clearance of a population of platelets refers to theremoval of a population of platelets from a unit volume of blood orserum per unit of time. Reducing the clearance of a population ofplatelets refers to preventing, delaying, or reducing the clearance ofthe population of platelets. Reducing clearance of platelets also maymean reducing the rate of platelet clearance.

Reducing the clearance of a platelet encompasses reducing clearance ofplatelets after storage at room temperature, or after chilling, as wellas “cold-induced platelet activation”. Cold-induced platelet activationis a term having a particular meaning to one of ordinary skill in theart. Cold-induced platelet activation may manifest by changes inplatelet morphology, some of which are similar to the changes thatresult following platelet activation by, for example, contact withglass. The structural changes indicative of cold-induced plateletactivation are most easily identified using techniques such as light orelectron microscopy. On a molecular level, cold-induced plateletactivation results in actin bundle formation and a subsequent increasein the concentration of intracellular calcium. Actin-bundle formation isdetected using, for example, electron microscopy. An increase inintracellular calcium concentration is determined, for example, byemploying fluorescent intracellular calcium chelators. Many of theabove-described chelators for inhibiting actin filament severing arealso useful for determining the concentration of intracellular calcium(Tsien, R., 1980, supra.). Accordingly, various techniques are availableto determine whether or not platelets have experienced cold-inducedactivation.

The effect of galactose incorporation on platelet clearance can bemeasured for example using either an in vitro system employingdifferentiated THP-1 cells or murine macrophages, isolated from theperitoneal cavity after thioglycolate injection stimulation. The rate ofclearance of modified platelets compared to unmodified platelets isdetermined. In these in vitro methods, galactose transfer is performedaccording to the protocol described above to modify platelets. Themodified platelets then are fed to the macrophages and ingestion of theplatelets by the macrophages is monitored. The in vitro assay shows ifgalactose transfer results in reduced ingestion of murine chilledplatelets, as it does for human platelets.

In some embodiments, the method for increasing circulation time of apopulation of platelets further comprises adding an enzyme thatcatalyzes the modification of a glycan moiety with a glycan modifyingagent. One example of an enzyme that catalyzes the modification of aglycan moiety is galactosyl transferase. Galactosyl transferasecatalyzes the glycation of the GP1bα molecule with UDP-galactose.

In some embodiments, the step of contacting the population of plateletswith at least one glycan modifying agent is performed in a platelet bag.

In accordance with the invention, the population of platelets can bechilled without the deleterious effects (cold-induced plateletactivation) usually experienced on chilling of platelets. The populationof platelets can be chilled prior to, concurrently with, or aftercontacting the platelets with the at least one glycan modifying agent.The selective modification of glycan moieties reduces clearance,following chilling (also if not chilled), thus permitting longer-termstorage than is presently possible. As used herein, chilling refers tolowering the temperature of the population of platelets to a temperaturethat is less than about 22° C. In some embodiments, the platelets arechilled to a temperature that is less than about 15° C. In somepreferred embodiments, the platelets are chilled to a temperatureranging from between about 0° C. to about 4° C.

In some embodiments, the population of platelets are stored chilled forat least 3 days. In some embodiments, the population of platelets arestored chilled for at least 5, 7, 10, 14, 21, and 28 days.

In some embodiments of the invention, the circulation time of thepopulation of platelets is increased by at least about 10%. In someother embodiments, the circulation time of the population of plateletsis increased by at least about 25%. In yet some other embodiments, thecirculation time of the population of platelets is increased by at leastabout 50%. In still yet other embodiments, the circulation time of thepopulation of platelets is increased by about 100%. As used herein,circulation time of a population of platelets is defined as the timewhen one-half of the platelets are no longer circulating.

The invention also embraces a method for increasing the storage time ofplatelets. As used herein the storage time of platelets is defined asthe time that platelets can be stored without substantial loss ofplatelet function or hemostatic activity such as the loss of the abilityto circulate. For example, ability to circulate after storage is reducedby less than about 1%, 2%, 3%, 5%, 10%, 20%, 30%, or 50%.

The platelets are collected from peripheral blood by standard techniquesknown to those of ordinary skill in the art. In some embodiments, theplatelets are contained in a pharmaceutically-acceptable carrier priorto treatment with a glycan modifying agent.

According to another aspect of the invention, a modified platelet or apopulation of modified platelets is provided. The modified plateletcomprises a plurality of modified glycan molecules on the surface of theplatelet. In some embodiments, the modified glycan moieties may bemoieties of GP1bα molecules. The modified glycan molecules comprise atleast one added sugar molecule. The added sugar may be a natural sugaror may be a non-natural sugar.

The invention also encompasses a platelet composition in a storagemedium. In some embodiments the storage medium comprises apharmaceutically acceptable carrier.

The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe platelets and that is a non-toxic material that is compatible with abiological system such as a cell, cell culture, tissue, or organism.Pharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

The invention further embraces a method for making a pharmaceuticalcomposition for administration to a mammal. The method comprisespreparing the above-described platelet preparation, and warming theplatelet preparation. In some embodiments, the method comprisesneutralizing the glycan modifying agent(s) and/or the enzyme(s) thatcatalyze the modification of the glycan moiety and placing theneutralized platelet preparation in a pharmaceutically acceptablecarrier. In a preferred embodiment, the chilled platelets are warmed toroom temperature (about 22° C.) prior to neutralization. In someembodiments, the platelets are contained in a pharmaceuticallyacceptable carrier prior to contact with the glycan modifying agent(s)with or without the enzyme(s) that catalyze the modification of theglycan moiety and it is not necessary to place the platelet preparationin a pharmaceutically acceptable carrier following neutralization.

As used herein, the terms “neutralize” or “neutralization” refer to aprocess by which the glycan modifying agent(s) and/or the enzyme(s) thatcatalyze the modification of the glycan moiety are renderedsubstantially incapable of glycan modification of the glycan residues onthe platelets. In some embodiments, the chilled platelets areneutralized by dilution, e.g., with a suspension of red blood cells.Alternatively, the treated platelets can be infused into the recipient,which is equivalent to dilution into a red blood cell suspension. Thismethod of neutralization advantageously maintains a closed system andminimizes damage to the platelets. In a preferred embodiment of glycanmodifying agents, no neutralization is required

An alternative method to reduce toxicity is by inserting a filter in theinfusion line, the filter containing, e.g. activated charcoal or animmobilized antibody, to remove the glycan modifying agent(s) and/or theenzyme(s) that catalyze the modification of the glycan moiety.

Either or both of the glycan modifying agent(s) and the enzyme(s) thatcatalyze the modification of the glycan moiety also may be removed orsubstantially diluted by washing the modified platelets in accordancewith standard clinical cell washing techniques.

The invention further provides a method for mediating hemostasis in amammal. The method includes administering the above-describedpharmaceutical preparation to the mammal. Administration of the modifiedplatelets may be in accordance with standard methods known in the art.According to one embodiment, a human patient is transfused with redblood cells before, after or during administration of the modifiedplatelets. The red blood cell transfusion serves to dilute theadministered, modified platelets, thereby neutralizing the glycanmodifying agent(s) and the enzyme(s) that catalyze the modification ofthe glycan moiety.

The dosage regimen for mediating hemostasis is selected in accordancewith a variety of factors, including the type, age, weight, sex andmedical condition of the subject, the severity of the disease, the routeand frequency of administration. An ordinarily skilled physician orclinician can readily determine and prescribe the effective amount ofplatelets required to mediate hemostasis.

The dosage regimen can be determined, for example, by following theresponse to the treatment in terms clinical signs and laboratory tests.Examples of such clinical signs and laboratory tests are well known inthe art and are described Harrison's Principles of Internal Medicine,15th Ed., Fauci A S et al., eds., McGraw-Hill, New York, 2001.

Also within the scope of the invention are storage compositions andpharmaceutical compositions for mediating hemostasis. In one embodiment,the compositions comprise a pharmaceutically-acceptable carrier, aplurality of modified platelets, a plurality of glycan modifyingagent(s) and optionally the enzyme(s) that catalyze the modification ofthe glycan moiety. The glycan modifying agent(s) and the enzyme(s) thatcatalyze the modification of the glycan moiety are present in thecomposition in sufficient amounts so as to reduce platelet clearance.Preferably, glycan modifying agent(s) (and optionally the enzyme(s) thatcatalyze the modification of the glycan moiety) are present in amountswhereby after chilling and neutralization, the platelets maintainsubstantially normal hemostatic activity. The amounts of glycanmodifying agent(s) (and optionally the enzyme(s) that catalyze themodification of the glycan moiety) which reduce platelet clearance canbe selected by exposing a preparation of platelets to increasing amountsof these agents, exposing the treated platelets to a chillingtemperature and determining (e.g., by microscopy) whether or notcold-induced platelet activation has occurred. Preferably, the amountsof glycan modifying agent(s) and the enzyme(s) that catalyze themodification of the glycan moiety can be determined functionally byexposing the platelets to varying amounts of glycan modifying agent(s)and the enzyme(s) that catalyze the modification of the glycan moiety,chilling the platelets as described herein, warming the treated(chilled) platelets, optionally neutralizing the platelets and testingthe platelets in a hemostatic activity assay to determine whether thetreated platelets have maintained substantially normal hemostaticactivity.

For example, to determine the optimal concentrations and conditions forpreventing cold-induced activation by a glycan modifying agent(s) (andoptionally the enzyme(s) that catalyze the modification of the glycanmoiety), increasing amounts of these agents are contacted with theplatelets prior to exposing the platelets to a chilling temperature. Theoptimal concentrations of the glycan modifying agent(s) and theenzyme(s) that catalyze the modification of the glycan moiety are theminimal effective concentrations that preserve intact platelet functionas determined by in vitro tests (e.g., observing morphological changesin response to glass, thrombin, cryopreservation temperatures;ADP-induced aggregation) followed by in vivo tests indicative ofhemostatic function (e.g., recovery, survival and shortening of bleedingtime in a thrombocytopenic animal or recovery and survival of⁵¹Cr-labeled platelets in human subjects).

According to yet another aspect of the invention, a composition foraddition to platelets to reduce platelet clearance or to increaseplatelet storage time is provided. The composition includes one or moreglycan modifying agents. In certain embodiments, the composition alsoincludes an enzyme(s) that catalyze the modification of the glycanmoiety. The glycan modifying agent and the enzyme(s) that catalyzes themodification of the glycan moiety are present in the composition inamounts that prevent cold-induced platelet activation. In one preferredembodiment, the glycan modifying agent is UDP-galactose and theenzyme(s) that catalyze the modification of the glycan moiety isgalactosyl transferase.

The invention also embraces a storage composition for preservingplatelets. The storage composition comprises at least one glycanmodifying agent in an amount sufficient to reduce platelet clearance. Insome embodiments the storage composition further comprises an enzymethat catalyzes the modification of a glycan moiety on the platelet. Theglycan modifying agent is added to the population of platelets that arepreferably kept between about room temperature and 37° C. In someembodiments, following treatment, the population of platelets is cooledto about 4° C. In some embodiments, the platelets are collected into aplatelet pack, bag, or container according to standard methods known toone of skill in the art. Typically, blood from a donor is drawn into aprimary container which may be joined to at least one satellitecontainer, all of which containers are connected and sterilized beforeuse. In some embodiments, the satellite container is connected to thecontainer for collecting platelets by a breakable seal. In someembodiments, the primary container further comprises plasma containing aplurality of platelets.

In some embodiments, the platelets are concentrated (e.g. bycentrifugation) and the plasma and red blood cells are drawn off intoseparate satellite bags (to avoid modification of these clinicallyvaluable fractions) prior to adding the glycan modifying agent with orwithout the enzyme that catalyzes the modification of a glycan moiety onthe platelet. Platelet concentration prior to treatment also mayminimize the amounts of glycan modifying agents required for reducingthe platelet clearance, thereby minimizing the amounts of these agentsthat are eventually infused into the patient.

In one embodiment, the glycan modifying agent(s) are contacted with theplatelets in a closed system, e.g. a sterile, sealed platelet pack, soas to avoid microbial contamination. Typically, a venipuncture conduitis the only opening in the pack during platelet procurement ortransfusion. Accordingly, to maintain a closed system during treatmentof the platelets with the glycan modifying agent(s), the agent(s) isplaced in a relatively small, sterile container which is attached to theplatelet pack by a sterile connection tube (see e.g., U.S. Pat. No.4,412,835, the contents of which are incorporated herein by reference).The connection tube may be reversibly sealed, or have a breakable seal,as will be known to those of skill in the art. After the platelets areconcentrated, e.g. by allowing the platelets to settle and squeezing theplasma out of the primary pack and into a second bag according tostandard practice, the seal to the container(s) including the glycanmodifying agent(s) is opened and the agents are introduced into theplatelet pack. In one embodiment, the glycan modifying agents arecontained in separate containers having separate resealable connectiontubes to permit the sequential addition of the glycan modifying agentsto the platelet concentrate.

Following contact with the glycan modifying agent(s), the treatedplatelets are chilled. In contrast to platelets stored at, for example,22° C., platelets stored at cryopreservation temperatures havesubstantially reduced metabolic activity. Thus, platelets stored at 4°C. are metabolically less active and therefore do not generate largeamounts of CO₂ compared with platelets stored at, for example, 22° C.(Slichter, S. J., 1981, Vox Sang 40 (Suppl 1), pp 72-86, ClinicalTesting and Laboratory-Clinical correlations.). Dissolution of CO₂ inthe platelet matrix results in a reduction in pH and a concommittantreduction in platelet viability (Slichter, S., 1981, supra.).Accordingly, conventional platelet packs are formed of materials thatare designed and constructed of a sufficiently permeable material tomaximize gas transport into and out of the pack (O₂ in and CO₂ out). Theprior art limitations in platelet pack design and construction areobviated by the instant invention, which permits storage of platelets atcryopreservation temperatures, thereby substantially reducing plateletmetabolism and diminishing the amount of CO₂ generated by the plateletsduring storage. Accordingly, the invention further provides plateletcontainers that are substantially non-permeable to CO₂ and/or CO₂ whichcontainers are useful particularly for cold storage of platelets.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES Example 1

Introduction

Modest cooling primes platelets for activation, but refrigeration causesshape changes and rapid clearance, compromising storage of platelets fortherapeutic transfusions. We found that shape change inhibition does notnormalize cold-induced clearance. We also found that cooling plateletsrearranges the surface configuration of the von Willebrand factor (vWf)receptor complex α subunit (GPIbα) such that it becomes targeted forrecognition by complement receptor 3 receptors (CR3) predominantlyexpressed on liver macrophages, leading to platelet phagocytosis andclearance. GPIb α removal prolongs survival of unchilled platelets.Chilled platelets bind vWf and function normally in vitro and ex vivoafter transfusion into CR3-deficient mice. Cooled platelets, however,are not “activated” like platelets exposed to thrombin or ADP, and theirvWf-receptor complex reacts normally with activated vWf.

As the temperature falls below 37° C. platelets become more susceptibleto activation by thrombotic stimuli, a phenomenon known as “priming”(Faraday and Rosenfeld, 1998; Hoffmeister et al., 2001). Priming may bean adaptation to limit bleeding at lower temperatures of body surfaceswhere most injuries occur. We propose that the hepatic clearancesystem's purpose is to remove repeatedly primed platelets, and thatconformational changes in GPIbα that promote this clearance do notaffect GPIbα's hemostatically important binding to vWf. Therefore,selective modification of GPIbα may accommodate cold storage ofplatelets for transfusion.

Materials and Methods

We obtained fluorescein isothiocyanate (FITC)-conjugated annexin V,phycoerythrin (PE)-conjugated anti-human CD11b/Mac-1 monoclonalantibodies (mAb), FITC-conjugated anti-mouse and anti-human IgM mAb,FITC-conjugated anti-mouse and anti-human CD62P-FITC mAb from Pharmingen(San Diego, Calif.); FITC-conjugated rat anti-mouse anti-human IgG mAbfrom Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.);FITC-conjugated anti-human CD61 mAbs (clone BL-E6) from AccurateScientific Corp. (Westbury, N.Y.); FITC-conjugated anti-human GPIbα mAb(clone SZ2) from Immunotech (Marseille, France); and FITC-conjugatedpolyclonal rabbit anti-vWf antibody from DAKOCytomation (Glostrup,Denmark). We purchased EGTA-acetoxymethylester (AM), Oregon Greencoupled fibrinogen from human plasma, CellTracker™ Orange CMTMR;CellTracker Green CMFDA, Nile-red (535/575) coupled andcarboxylate-modified 1 μm microspheres/FluoSpheres from MolecularProbes, Inc. (Eugene, Oreg.) and ¹¹¹Indium from NEN Life ScienceProducts (Boston, Mass.). We purchased Cytochalasin B, dimethylsulfoxide (DMSO), trisodium isothiocyanate (TRITC), human thrombin,prostaglandin E1 (PGE₁), phorbol ester 12-tetradecanoylphorbol-13acetate (PMA), A23187 ionophore from Sigma (St. Louis, Mo.); botrocetinfrom Centerchem Inc. (Norwalk, Conn.); andO-sialoglycoprotein-endopeptidase from Cerladane (Hornby, Canada). HBSScontaining Ca²⁺ and Mg²⁺, pH 6.4; RPMI 1640; 0.05% Trypsin-EDTA (0.53mM) in HBSS without Ca²⁺ and Mg²⁺; and other supplements (penicillin,streptomycin and fetal bovine serum) were from GIBCO Invitrogen Corp.(Grand Island, N.Y.). TGF-β1 from Oncogene Research Products (Cambridge,Mass.); 1,25-(OH)₂ vitamin D3 from Calbiochem (San Diego, Calif.); andAdenosine-5′-Diphosphate (ADP) were from USB (Cleveland, Ohio). Avertin(2,2,2-tribromoethanol) was purchased from Fluka Chemie (Steinheim,Germany). Collagen related peptide (CRP) was synthesized at the TuftsCore Facility, Physiology Dept. (Boston, Mass.) and cross-linked aspreviously described (Morton et al., 1995). Mocarhagin, a snake venommetalloprotease, was provided by Dr. M. Berndt, Baker Medical ResearchInstitute, Melbourne Victoria 318 1, Australia. Additional unconjugatedanti mouse GPIbα mAbs and a PE-conjugated anti-mouse GPIbα mAb pOp4 wereprovided by Dr. B. Nieswandt (Witten/Herdecke University, Wuppertal,Germany). We obtained THP-1 cells from the American Type CultureCollection (Manassas, Va.).

Animals

For assays of clearance and survival studies, we used age-, strain- andsex-matched C57BL/6 and C57BL/6×129/sv wild type mice obtained fromJackson Laboratory (Bar Harbor, Me.). C57BL/6×129/sv mice deficient incomplement component C3 (Wessels et al., 1995) were provided by Dr. M.C. Carroll (Center for Blood Research and Department of Pediatrics,Harvard Medical School, Boston, Mass.). C57BL/6 mice deficient in CR3(Coxon et al., 1996) were provided by Dr. T Mayadas and C57BL/6 micedeficient in vWf (Denis et al., 1998) were provided by Dr. D. Wagner.Mice were maintained and treated as approved by Harvard Medical AreaStanding Committee on Animals according to NIH standards as set forth inThe Guide for the Care and Use of Laboratory Animals.

Human Platelets

Blood was drawn from consenting normal human volunteers (approval wasobtained from the Institutional Review Boards of both Brigham andWomen's Hospital and the Center for Blood Research (Harvard MedicalSchool)) by venipuncture into 0.1 volume of Aster-Jandl citrate-basedanticoagulant (Hartwig and DeSisto, 1991) and platelet rich plasma (PRP)was prepared by centrifugation of the anticoagulated blood at 300×g for20 min at room temperature. Platelets were separated from plasmaproteins by gel-filtration at room temperature through a small Sepharose2B column (Hoffmeister et al., 2001). Platelets used in the in vitrophagocytosis assay described below were labeled with 1.8 μM CellTracker™Orange CMTMR (CM-Orange) for 20 min at 37° C. (Brown et al., 2000), andunincorporated dye was removed by centrifugation (850×g, 5 min.) with 5volumes of washing buffer containing 140 mM NaCl, 5 mM KCl, 12 mMtrisodium citrate, 10 mM glucose, and 12.5 mM sucrose, 1 μg/ml PGE₁, pH6.0 (buffer A). Platelets were resuspended at 3×10⁸/ml in a solutioncontaining 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl₂, 5 mM NaHCO₃, 10 mMglucose and 10 mM Hepes, pH 7.4 (buffer B).

The N-terminus of GPIbα was enzymatically removed from the surface ofchilled or room temperature maintained and labeled platelets in bufferB, also containing 1 mM Ca²⁺ and 10 μg/ml of the snake venommetalloprotease mocarhagin (Ward et al., 1996). After the enzymaticdigestion, the platelets were washed by centrifugation with 5× volume ofbuffer A and routinely checked by microscopy for aggregates.GPIbα-N-terminus removal was monitored by incubating plateletsuspensions with 5 μg/ml of FITC-conjugated anti-human GPIbα (SZ2) mAbfor 10 min at room temperature and followed by immediate flow cytometryanalysis on a FACScalibur Flow Cytometer (Becton Dickinson Biosciences,San Jose, Calif.). Platelets were gated by forward/side scattercharacteristics and 50,000 events acquired.

Murine Platelets

Mice were anesthetized with 3.75 mg/g (2.5%) of Avertin, and 1 ml bloodwas obtained from the retroorbital eye plexus into 0.1 volume ofAster-Jandl anticoagulant. PRP was prepared by centrifugation ofanticoagulated blood at 300×g for 8 min at room temperature. Plateletswere separated from plasma proteins by centrifugation at 1200×g for 5min and washed two times by centrifugation (1200×g for 5 min) using 5×volumes of washing buffer (buffer A). This procedure is meant bysubsequent use of the term “washed”. Platelets were resuspended at aconcentration of 1×10⁹/ml in a solution containing 140 mM NaCl, 3 mMKCl, 0.5 mM MgCl₂, 5 mM NaHCO₃, 10 mM glucose and 10 mM Hepes, pH 7.4(buffer B). Platelet count was determined using a Bright LineHemocytometer (Hausser Scientific, Horsham, Pa.) under a phase-contrastmicroscope at 400× magnification. Some radioactive platelet clearancestudies were performed with ¹¹¹Indium, and we labeled mouse plateletsusing a method described for primate platelets (Kotze et al., 1985).Platelets were resuspended at a concentration of 2×10⁹/ml in 0.9% NaCl,pH 6.5 (adjusted with 0.1 M sodium citrate), followed by the addition of500 μCi ¹¹¹Indium chloride for 30 min at 37° C. and washed as describedabove and suspended in buffer B at a concentration of 1×10⁹/ml.

For intravital microscopy or other platelet survival experiments, washedplatelets were labeled either with 2.5 μM CellTracker Green CMFDA(5-chloromethyl fluorescein diacetate) (CMFDA) for 20 min at 37° C.(Baker et al., 1997) or with 0.15 μM TRITC for 20 min at 37° C. Inbuffer B also containing 0.001% DMSO, 20 mM HEPES. Unincorporated dyewas removed by centrifugation as described above, and platelets weresuspended at a concentration of 1×10⁹/ml in buffer B.

The N-terminus of GPIbα was enzymatically removed from the surface ofchilled or room temperature labeled platelets with 100 μg/mlO-sialoglycoprotein endopeptidase in buffer B containing 1 mM Ca²⁺ for20 min at 37° C. (Bergmeier et al., 2001). After enzymatic digestion,platelets were washed by centrifugation and checked by light microscopyfor aggregates. Enzymatic removal of the GPIbα-N-terminus removal wasmonitored by incubating the platelet suspensions with 5 μg/ml ofPE-conjugated anti-mouse GPIbα mAb pOp4 for 10 min at room temperature,and bound PE analyzed by flow cytometry.

To inhibit cold-induced platelet shape changes, 10⁹/ml platelets inbuffer B were loaded with 2 μM EGTA-AM followed by 2 μM cytochalasin Bas previously described (Winokur and Hartwig, 1995), labeled with 2.5 μMCMFDA for 30 min at 37° C. and then chilled or maintained at roomtemperature. The platelets were subjected to standard washing andsuspended at a concentration of 1×10⁹/ml in buffer B before injectioninto mice.

Platelet Temperature Protocols

To study the effects of temperature on platelet survival or function,unlabeled, radioactively labeled, or fluorescently-labeled mouse orhuman platelets were incubated for 2 hours at room temperature (25-27°C.) or else at ice bath temperatures and then rewarmed for 15 minutes at37° C. before transfusion into mice or in vitro analysis. Plateletssubjected to these treatments are designated cooled or chilled (orchilled, rewarmed) and room temperature platelets respectively.

Murine Platelet Recovery, Survival and Fate

CMFDA labeled chilled or room temperature murine platelets (10⁸) wereinjected into syngeneic mice via the lateral tail vein using a 27-gaugeneedle. For recovery and survival determination, blood samples werecollected immediately (<2 min) and 0.5, 2, 24, 48, 72 hours aftertransfusion into 0.1 volume of Aster-Jandl anticoagulant. Whole bloodanalysis using flow cytometry was performed and the percentage of CMFDApositive platelets determined by gating on all platelets according totheir forward and side scatter characteristics (Baker et al., 1997).50,000 events were collected in each sample. CMFDA positive plateletsmeasured at a time <2 min was set as 100%. The input of transfusedplatelets per mouse was ˜2.5-3% of the whole platelet population.

To evaluate the fate of platelets, tissues (heart, lung, liver, spleen,mucle, and femur) were harvested at 0.5, 1 and 24 hours after theinjection of 10⁸ chilled or room temperature ¹¹¹Indium labeled plateletsinto mice. The organ-weight and their radioactivity were determinedusing a Wallac 1470 Wizard automatic gamma counter (Wallac Inc.,Gaitersburg, Md.). The data were expressed as gamma count per gramorgan. For recovery and survival determination of radioactive platelets,blood samples were collected immediately (<2 min) and 0.5 and hoursafter transfusion into 0.1 volume of Aster-Jandl anticoagulant and theirgamma counts determined (Kotze et al., 1985).

Platelet Aggregation

Conventional tests were performed and monitored in a Bio/Dataaggregometer (Horsham, Pa.). Samples of 0.3-ml murine washed and stirredplatelets were exposed to 1 U/ml thrombin, 10 μM ADP, or 3 μg/ml CRP at37° C. Light transmission was recorded over 3 min.

Activated VWf Binding

Platelet rich plasma was treated with or without 2 U/ml botrocetin for 5min at 37° C. (Bergmeier et al., 2001). Bound vWf was detected by flowcytometry using FITC conjugated polyconal rabbit anti-vWf antibody.

Surface Labeling of Platelet GPIbα

Resting mouse platelets maintained at room temperature or chilled 2 hrswere diluted to a concentration of 2×10⁶/ml in phosphate buffered saline(PBS) containing 0.05% glutaraldehyde. Platelet solutions (200 μl) wereplaced on a polylysine-coated glass coverslip contained in wells of96-well plate, and the platelets were adhered to each coverslip bycentrifugation at 1,500×g for 5 min at room temperature. The supernatantfluid was then removed, and platelets bound to the coverslip were fixedwith 0.5% glutaraldehyde in PBS for 10 min. The fixative was removed,unreacted aldehydes quenched with a solution containing 0.1% sodiumborohydride in PBS followed by washing with PBS containing 10% BSA.GPIbα on the platelet surface was labeled with a mixture of three ratanti-mouse GPIbα monoclonal antibodies, each at 10 μg/ml (Bergmeier etal., 2000) for 1 hr followed by 10 nm gold coated with goat anti-ratIgG. The coverslips were extensively washed with PBS, post-fixed with 1%glutaraldehyde, washed again with distilled water, rapidly frozen,freeze-dried, and rotary coated with 1.2 nm of platinum followed by 4 nmof carbon without rotation in a Cressington CFE-60 (Cressington,Watford, UK). Platelets were viewed at 100 kV in a JEOL 1200-EX electronmicroscope (Hartwig et al., 1996; Kovacsovics and Hartwig, 1996)

In Vitro Phagocytic Assay

Monocytic THP-1 cells were cultured for 7 days in RPMI 1640 cell culturemedia supplemented with 10% fetal bovine serum, 25 mM Hepes, 2 mMglutamine and differentiated using 1 ng/ml TGFP and 50 nM 1,25-(OH)₂vitamin D3 for 24 hours, which is accompanied by increased expression ofCR3 (Simon et al., 2000). CR3 expression was monitored by flow cytometryusing a PE-conjugated anti-human CD11b/Mac-1 mAb. Undifferentiated ordifferentiated THP-1 cells (2×10⁶/ml) were plated onto 24-well platesand allowed to adhere for 45 minutes at 37° C. The adherentundifferentiated or differentiated macrophages were activated by theaddition of 15 ng/ml PMA for 15 min. CM-range-labeled, chilled or roomtemperature platelets (10⁷/well), previously subjected to differenttreatments were added to the undifferentated or differentiatedphagocytes in Ca²⁺— and Mg²⁺-containing HBSS and incubated for 30 min at37° C. Following the incubation period, the phagocyte monolayer waswashed with HBSS for 3 times, and adherent platelets were removed bytreatment with 0.05% trypsin/0.53 mM EDTA in HBSS at 37° C. for 5 minfollowed by 5 mM EDTA at 4° C. to detach the macrophages for flowcytometric analysis of adhesion or ingestion of platelets (Brown et al.,2000). Human CM-Orange-labeled, chilled or room temperature plateletsall expressed the same amount of the platelet specific marker CD61 asfreshly isolated unlabeled platelets (not shown). CM-Orange-labeledplatelets incubated with macrophages were resolved from the phagocytesaccording to their forward and side scatter properties. The macrophageswere gated, 10,000 events acquired for each sample, and data analyzedwith CELLQuest software (Becton Dickenson). CM-Orange-labeled plateletsthat associate with the phagocyte population have a shift in orangefluorescence (FIG. 6 a and FIG. 6 b, ingested, y axis). These plateletswere ingested rather than merely adherent, because they failed to duallabel with the FITC-conjugated mAb to CD61.

Immunolabeling and Flow Cytometry of Platelets

Washed murine or human platelets (2×10⁶) were analyzed for surfaceexpression of CD62P, CD61, or surface bound IgM and IgG after chillingor room temperature storage by staining with fluorophore-conjugated Abs(5 μg/ml) for 10 min at 37° C. Phosphatidylserine exposure by chilled orroom temperature platelets was determined by resuspending 5 μl ofplatelets in 400 μl of HBSS containing 10 mM Ca²⁺ with 10 μg/ml ofFITC-conjugated annexin-V. As a positive control for PS exposure,platelet suspensions were stimulated with 1 μM A23187. Fibrinogenbinding was determined by the addition of Oregon Green-fibrinogen for 20min at room temperature. All platelet samples were analyzed immediatelyby flow cytometry. Platelets were gated by forward and side scattercharacteristics.

Intravital Microscopy Experiments

Animal preparation, technical and experimental aspects of the intravitalvideo microscopy setup have been described (von Andrian, 1996). Six toeight week-old mice of both sexes were anesthetized by intraperitonealinjection of a mixture of Xylazine and Ketamin. The right jugular veinwas catheterized with PE-10 polyethylene tubing. The lower surface ofthe left liver lobe was surgically prepared and covered by a glass coverslip for further in vivo microscopy as described (McCuskey, 1986). 10⁸chilled platelets and room temperature platelets labeled with CMFDA andTRITC respectively were mixed 1:1 and administered intravenously. Thecirculation of labeled platelets in liver sinusoids was followed byvideo triggered stroboscopic epi-illumination. Ten video scenes wererecorded from 3 centrilobular zones at each indicated time point. Theratio of cooled (CMFDA)/RT (TRITC) adherent platelets in the identicalvisualized field was calculated. Confocal microscopy was performed usinga Radiance 2000 MP confocal-multiphoton imaging system connected to anOlympus BX 50 WJ upright microscope (Biorad, Hercules, Calif.), using a10× water immersion objective. Images were captured and analyzed withLaser Sharp 2000 software (Biorad) (von Andrian, 2002).

Platelet Aggregation in Shed Blood

We used a flow cytometric method to analyze aggregate formation byplatelets in whole blood emerging from a wound as described for primates(Michelson et al., 1994). We injected 10⁸ CMFDA labeled room temperaturemurine platelets into syngeneic wild type mice and 10⁸ CMFDA labeled,chilled platelets into CR3-deficient mice. Twenty-four hours after theplatelet infusion, a standard bleeding time assay was performed,severing a 3-mm segment of a mouse tail (Denis et al., 1998). Theamputated tail was immersed in 100 μl 0.9% isotonic saline at 37° C. Theemerging blood was collected for 2 min., and 0.1 volume of Aster-Jandlanticoagulant added and followed immediately with 1% paraformaldehyde(final concentration). Peripheral blood was obtained by retroorbital eyeplexus bleeding in parallel as described above and immediately fixedwith 1% paraformaldehyde (final concentration). To analyze the number ofaggregates in vivo by flow cytometry, the shed blood emerging from thebleeding time wound, as well as a peripheral whole blood sample, werediluted and labeled with PE-conjugated anti-murine GPIbα mAb pOp4 (5μg/ml, 10 min.). Platelets were discriminated from red cells and whitecells by gating according to their forward scatter characteristics andGPIbα positivity. A histogram of log forward light scatter (reflectingplatelet size) versus GPIbα binding was then generated. In theperipheral whole blood samples, analysis regions were plotted around theGPIbα-positive particles to include 95% of the population on the forwardscatter axis (region 1) and the 5% of particles appearing above thisforward light scatter threshold (region 2). Identical regions were usedfor the shed blood samples. The number of platelet aggregates in shedblood as a percentage of the number of single platelets was calculatedfrom the following formula: [(number of particles in region 2 of shedblood)−(number of particles in region 2 of peripheral blood)]÷(number ofparticles in region 1 of shed blood)×100%. The infused platelets wereidentified by their CMFDA labeling and discriminated from the CMFDAnegative non-infused platelets.

Flow Cytometric Analysis of Murine Platelet Fibrinogen Binding andP-selectin Exposure of Circulating Platelets

Room temperature CM-Orange-labeled room temperature platelets (10⁸) wereinjected into wild type mice and CM-Orange-chilled labeled platelets(10⁸) into CR3 deficient mice. Twenty-four hours after platelet infusionthe mice were bled and the platelets isolated. Resting or thrombinactivated (1 U/ml, 5 min) platelet suspensions (2×10⁸) were diluted inPBS and either stained with FITC-conjugated anti-mouse P-selectin mAb orwith 50 μg/ml Oregon Green-conjugated fibrinogen for 20 min at roomtemperature. Platelet samples were analyzed immediately by flowcytometry. Transfused and non-transfused platelets were gated by theirforward scatter and CM-Orange fluorescence characteristics. P-selectinexpression and fibrinogen binding were measured for each CM-Orangepositive and negative population before and after stimulation withthrombin.

Statistics

The intravital microscopy data are expressed as means±SEM. Groups werecompared using the nonpaired t test. P values<0.05 were consideredsignificant. All other data are presented as the mean±SD.

Results

The Clearance of Chilled Platelets Occurs Predominantly in the Liver andis Independent of Platelet Shape.

Mouse platelets kept at room temperature (RT) and infused into syngeneicmice disappear at fairly constant rate over time for about 80 hours(FIG. 1A). In contrast, approximately two-thirds of mouse plateletschilled at ice-bath temperature and rewarmed (Cold) before injectionrapidly disappear from the circulation as observed previously in humansand mice (Becker et al., 1973; Berger et al., 1998). Chilled andrewarmed platelets treated with the cell-permeable calcium chelatorEGTA-AM and the actin filament barbed end capping agent cytochalasin B(Cold+CytoB/EGTA) to preserve their discoid shape (Winokur and Hartwig,1995), left the circulation as rapidly as chilled, untreated plateletsdespite the fact that these platelets were fully functional asdetermined by thrombin-, ADP- or collagen related peptide-(CRP) inducedaggregation in vitro (FIG. 1B). The recoveries of infused plateletsimmediately following transfusion were 50-70%, and the kinetics ofplatelet disappearance were indistinguishable whether we used ¹¹¹Indiumor CMFDA to label platelets. The relative survival rates of roomtemperature and chilled mouse platelets resemble the values reportedpreviously for identically treated mouse (Berger et al., 1998) and humanplatelets (Becker et al., 1973).

FIG. 1C shows that the organ destinations of room temperature andchilled mouse platelets differ. Whereas room-temperature plateletsprimarily end up in the spleen, the liver is the major residence ofchilled platelets removed from the circulation. A greater fraction ofradionuclide detected in the kidneys of animals receiving¹¹¹Indium-labeled chilled compared with room-temperature platelets at 24hours may reflect a more rapid degradation of chilled platelets anddelivery of free radionuclide to the urinary system. One hour afterinjection the organ distribution of platelets labeled with CMFDA wascomparable to that of platelets labeled with ¹¹¹Indium. In both cases,60-90% of the labeled chilled platelet population deposited in theliver, ˜20% in the spleen and ˜15% in the lung. In contrast, a quarterof the infused room temperature platelets distributed equally among theliver, spleen and lung.

Chilled Platelets Co-localize with Liver Macrophages (Kupffer Cells).

The clearance of chilled platelets by the liver and the evidence forplatelet degradation is consistent with recognition and ingestion ofchilled platelets by Kupffer cells, the major phagocytic scavenger cellsof the liver. FIG. 1D shows the location of phagocytotic Kupffer cellsand adherent chilled CMFDA-labeled platelets in a representativeconfocal micrograph of a mouse liver section 1 hour after transfusion.Sinusoidal macrophages were visualized by the injection of 1 μm carboxylmodified polystyrene microspheres marked with Nile-red. Co-localizationof transfused platelets and macrophages is indicated in yellow in themerged micrograph of both fluorescence emissions. The chilled plateletslocalize with Nile-red-labeled cells preferentially in the periportaland midzonal domains of liver acini, sites rich in sinusoidalmacrophages (Bioulac-Sage et al., 1996; MacPhee et al., 1992).

CR3-deficient Mice do not Rapidly Clear Chilled Platelets.

CR3 (α_(M)β₂ integrin; CD11b/CD18; Mac-1) is a major mediator ofantibody independent clearance by hepatic macrophages. FIG. 2 a showsthat chilled platelets circulate in CR3-deficient animals with the samekinetics as room-temperature platelets, although the clearance of bothplatelet populations is shorter in the CR3-deficient mouse compared tothat in wild-type mice (FIG. 1 a). The reason for the slightly fasterplatelet removal rate by CR3-deficient mice compared to wild-type miceis unclear. Chilled and rewarmed platelets also clear rapidly fromcomplement factor 3 C3-deficient mice (FIG. 2 c), missing a majoropsonin that promotes phagocytosis and clearance via CR3 and from vonWillebrand factor (vWf) deficient mice (Denis et al., 1998) (FIG. 2 b).

Chilled Platelets Adhere Tightly to Kupffer Cells in vivo.

Platelet adhesion to wild-type liver sinusoids was further investigatedby intravital microscopy, and the ratio between chilled and roomtemperature stored adherent platelets infused together was determined.FIG. 3 shows that both chilled and room temperature platelets attach tosinusoidal regions with high Kupffer cell density (FIGS. 3 a and 3 b),but that 2.5 to 4-times more chilled platelets attach to Kupffer cellsin the wild-type mouse than room-temperature platelets (FIG. 3 c). Incontrast, the number of platelets adhering to Kupffer cells inCR3-deficient mice was independent of chilling or room temperatureexposure (FIG. 3 c).

Chilled Platelets Lacking the N-terminal Domain of GPIbα CirculateNormally.

Because GPIbα, a component of the GPIb-IX-V receptor complex for vWf,can bind CR3 under certain conditions in vitro (Simon et al., 2000), weinvestigated GPIbα as a possible counter receptor on chilled plateletsfor CR3. The O-sialoglycoprotein endopeptidase cleaves the 45-kDaN-terminal extracellular domain of the murine platelet GPIbα, leavingother platelet receptors such as (α_(IIb)β₃, α₂α₁, GPVI/FcRγ-chain andthe protease-activated receptors intact (Bergmeier et al., 2001). Hence,we stripped this portion of the extracellular domain of GPIbα from mouseplatelets with O-sialoglycoprotein endopeptidase (FIG. 4A inset) andexamined their survival in mice following room temperature or coldincubation. FIG. 4A shows that chilled platelets no longer exhibit rapidclearance after cleavage of GPIbα. In addition, GPIbα depleted roomtemperature-treated platelets have slightly elongated survival times(˜5-10%) when compared to the GPIbα-containing room-temperaturecontrols.

Chilling does not Affect Binding of Activated vWf to the PlateletvWf-receptor but Induces Clustering of GPIBα on the Platelet Surface.

FIG. 4B shows that botrocetin-activated vWf binds GPIbα equally well onroom temperature as on cold platelets, although chilling of plateletsleads to changes in the distribution of GPIbα on the murine plateletsurface. GPIbα molecules, identified by immunogold labeled monoclonalmurine anti-GPIbα antibodies, form linear aggregates on the smoothsurface of resting discoid platelets at room temperature (FIG. 4C, RT).This arrangement is consistent with information about the architectureof the resting blood platelet. The cytoplasmic domain of GPIbα bindslong filaments curving with the plane of the platelet membrane throughthe intermediacy of filamin A molecules (Hartwig and DeSisto, 1991).After chilling (FIG. 4C, Chilled) many GPIbα molecules organize asclusters over the platelet membrane deformed by internal actinrearrangements (Hoffmeister et al., 2001; Winokur and Hartwig, 1995).

Recognition of Platelet GPIbα by CR3-mediates Phagocytosis of ChilledHuman Platelets In Vitro.

Differentiation of human monocytoid THP-1 cells using TGF-β1 and1,25-(OH)₂ Vitamin D3 increases expression of CR3 by ˜2-fold (Simon etal., 1996). Chilling resulted in 3-fold increase of plateletphagocytosis by undifferentiated THP-1 cells and a ˜5-fold increase bydifferentiated THP-1 cells (FIGS. 5B and 5 c), consistent with mediationof platelet uptake by CR3. In contrast, the differentiation of THP-1cells had no significant effect on the uptake of room temperature storedplatelets (FIGS. 5A and 5 c). To determine if GPIbα is the counterreceptor for CR3-mediated phagocytosis on chilled human platelets, weused the snake venom metalloprotease mocarhagin, to remove theextracellular domain of GPIbα (Ward et al., 1996). Removal of humanGPIbα from the surface of human platelets with mocarhagin reduced theirphagocytosis after chilling by ˜98% (FIG. 5C).

Exclusion of Other Mediators of Cold-induced Platelet Clearance

Table 1 shows results of experiments that examined whether coolingaffected the expression of platelet receptors other than GPIbα or theirinteraction with ligands. These experiments revealed no detectableeffects on the expression of P-selectin, α_(IIb)β₃-integrin density oron α_(IIb)β₃ fibrinogen binding, a marker of α_(IIb)β₃ activation.Chilling also did not increase phosphatidylserine (PS) exposure, anindicator of apoptosis, nor did it change platelet binding of IgG or IgMimmunoglobulins.

TABLE 1 Effect of chilling on binding of various antibodies or ligandsto platelet receptors. Binding ratio 4° C.:22° C. Platelet receptor(ligand) Human platelets Murine platelets P-Selectin (anti-CD62P mAb)1.01 ± 0.06 1.02 ± 0.03 Platelet associated IgGs 1.05 ± 0.14 1.06 ± 0.03Platelet associated IgMs 0.93 ± 0.10 1.01 ± 0.02 Phosphatidylserine(annexin V) 0.95 ± 0.09 1.04 ± 0.02 α_(IIb)β₃ (anti-CD61 mAb) 1.03 ±0.05 1.04 ± 0.10 α_(IIb)β₃ (fibrinogen) 1.05 ± 0.10 1.06 ± 0.06

The binding of fluorescently labeled antibodies or ligands againstvarious receptors on chilled-rewarmed or room temperature human andmurine platelets was measured by flow cytometry. The data are expressedas the ratio between the mean fluophore bound to the surface of chilledversus room temperature platelets (mean±SD, n=3-4).

Circulating Chilled Platelets have Hemostatic Function in CR3-deficientMice.

Despite their rapid clearance in wild type mice, CM-Orange or CMFDAlabeled chilled platelets were functional 24 h after infusion intoCR3-deficient mice, as determined by three independent methods. First,chilled platelets incorporate into platelet aggregates in shed bloodemerging from a standardized tail vein bleeding wound (FIG. 6).CMFDA-positive room temperature platelets transfused into wild type mice(FIG. 6 b) and CNIFDA-positive chilled platelets transfused intoCR3-deficient mice (FIG. 6 d) formed aggregates in shed blood to thesame extent as CMFDA-negative platelets of the recipient mouse. Second,as determined by platelet surface exposure of the fibrinogen-bindingsite on α_(IIb)β₃ 24 hours after transfusion of CM-Orange-labeledchilled and rewarmed platelets into CR3 deficient mice following ex vivostimulation by thrombin. Third, CM-Orange platelets chilled and rewarmedwere fully capable of upregulation of P-selectin in response to thrombinactivation (FIG. 6 e).

Discussion

Cold-induced Platelet Shape Change Alone does not Lead to PlateletClearance In Vivo

Cooling rapidly induces extensive platelet shape changes mediated byintracellular cytoskeletal rearrangements (Hoffmeister et al., 2001;White and Krivit, 1967; Winokur and Hartwig, 1995). These alterationsare partially but not completely reversible by rewarming, and rewarmedplatelets are more spherical than discoid. The idea that preservation ofplatelet discoid shape is a major requirement for platelet survival hasbeen a dogma, despite evidence that transfused murine and baboonplatelets activated ex vivo by thrombin circulate normally withextensive shape changes (Berger et al., 1998; Michelson et al, 1996).Here we have shown that chilling leads to specific changes in theplatelet surface that mediate their removal independently of shapechange, and that the shape change per se does not lead to rapid plateletclearance. Chilled and rewarmed platelets, preserved as discs withpharmacological agents, clear with the same speed as untreated chilledplatelets, and misshapen chilled and rewarmed platelets circulate likeroom temperature maintained platelets in CR3-deficient mice. The smallsize of platelets may allow them to remain in the circulation, escapingentrapment despite these extensive shape deformities.

Receptors Mediating Clearance of Chilled Platelets: CR3 and GPIbα

The normal platelet life span in humans is approximately 7 days (Aas,1958; Ware et 2000). The incorporation of platelets into small bloodclots engendered by continuous mechanical stresses undoubtedlycontributes to platelet clearance, because massive clotting reactions,such as occur during disseminated intravascular coagulation, causethrombocytopenia (Seligsohn, 1995). The fate of platelets in suchclotting reactions differs from that of infused ex vivo-activatedplatelets such as in the experiments of Michelson et al (Michelson etal., 1996) and Berger et al (Berger et al., 1998), because in vivoplatelet stimulation occurs on injured vessel walls, and the activatedplatelets rapidly sequester at these sites.

Isoantibodies and autoantibodies accelerate the phagocytic removal ofplatelets by Fc-receptor-bearing macrophages in individuals sensitizedby immunologically incompatible platelets or in patients with autoimmunethrombocytopenia, but otherwise little information exists regardingmechanisms of platelet clearance. We showed, however, that thequantities of IgG or IgM bound to chilled or room-temperature humanplatelets are identical, implying that binding of platelet-associatedantibodies to Fc-receptors does not mediate the clearance of cooledplatelets. We also demonstrated that chilling of platelets does notinduce detectable phosphatidylserine (PS) exposure on the plateletsurface in vitro militating against PS exposure and the involvement ofscavenger receptors in the clearance of chilled platelets.

Although many publications have referred to effects of cold on plateletsas “activation”, aside from cytoskeletally-mediated shape changes,chilled platelets do not resemble platelets activated by stimuli such asthrombin or ADP. Normal activation markedly increases surface P-selectinexpression, a consequence of secretion from intracellular granules(Berman et al., 1986). Chilling of platelets does not lead toup-regulation of P-selectin (Table 1), but the clearance of chilledplatelets isolated from wild-type or P-selectin-deficient mice isequally rapid (Berger et al., 1998). Activation also increases theamount of α_(IIb)β₃-integrin and its avidity for fibrinogen (Shattil,1999), but cooling does not have these effects (Table 1). The normalsurvival of thrombin-activated platelets is consistent with ourfindings.

We have shown that CR3 on liver macrophages is primarily responsible forthe recognition and clearance of cooled platelets. The predominant roleof CR3 bearing macrophages in the liver in clearance of chilledplatelets despite abundant CR3-expressing macrophages in the spleen isconsistent with the principally hepatic clearance of IgM-coatederythrocytes (Yan et al., 2000) and may reflect blood filtrationproperties of the liver that favor binding and ingestion by macrophageCR3. The extracellular domain of GPIbα binds avidly to CR3, and undershear stress in vitro supports the rolling and firm adhesion of THP-1cells (Simon et al., 2000). Cleavage of the extracellular domain ofmurine GPIbα results in normal survival of chilled platelets transfusedinto mice. GPIbα depletion of human chilled platelets greatly reducesphagocytosis of the treated platelets by macrophage-like cells in vitro.We propose, therefore, that GPIbα is the co-receptor for livermacrophage CR3 on chilled platelets leading to platelet clearance byphagocytosis.

The normal clearance of cold platelets lacking the N-terminal portion ofGPIbα rules out the many other CR3-binding partners, including moleculesexpressed on platelet surfaces as candidates for mediating chilledplatelet clearance. These ligand candidates include ICAM-2, fibrinogenbound to the platelet integrin α_(IIb)β₃, iC3b, P-selectin,glucosaminoglycans, and high molecular weight kininogen. We excludeddeposition of the opsonic C3b fragment iC3b as a mechanism for chilledplatelet clearance using mice deficient in complement factor 3, and theexpression level of α_(IIb)β₃ and fibrinogen binding are also unchangedafter chilling of platelets.

Binding to Activated vWf and Cold-induced Binding to CR3 Appear to beSeparate Functions of GPIbα.

GPIbα on the surface of the resting discoid platelet exists in lineararrays (FIG. 5) in a complex with GPIbα, GPIX and V, attached to thesubmembrane actin cytoskeleton by filamin-A and Filamin B (Stossel etal., 2001). Its role in hemostasis is to bind the activated form of vWfat sites of vascular injury. GPIbα binding to activated vWf isconstitutive and requires no active contribution from the platelet,since activated vWf binds equally well to GPIbα on resting or onstimulated platelets. Stimulation of platelets in suspension by thrombinand other agonists causes GPIbα to redistribute in part from theplatelet surface into an internal membrane network, the open canalicularsystem, but does not lead to platelet clearance in vivo (Berger et al.,1998; Michelson et al., 1996) or to phagocytosis in vitro (unpublishedobservations). Cooling of platelets however, causes GPIbα clusteringrather than internalization. This clustering is independent of barbedend actin assembly, because it occurs in the presence of cytochalasin B.

Despite cold's promoting recognition of platelet GPIbα by CR3, it has noeffect on interaction between GPIbα and activated vWf in vitro, andchilled platelets transfused into vWf-deficient mice disappear asrapidly as in wild-type mice. The separability of GPIbα's interactionwith vWf and CR3 suggests that selective modification of GPIbα. mightinhibit cold-induced platelet clearance without impairment of GPIbα'shemostatically important reactivity with vWf. Since all tests ofplatelet function of cooled platelets in vitro and after infusion intoCR3-deficient mice yielded normal results, suitably modified plateletswould predictably be hemostatically effective.

Physiological Importance of Cold-induced Platelet Clearance.

Although gross platelet shape changes become obvious only attemperatures below 15° C., accurate biochemical analyses show thatcytoskeletal alterations and increased responsiveness to thrombin aredetectable as the temperature falls below 37° C. (Faraday and Rosenfeld,1998; Hoffmeister et al., 2001; Tablin et al., 1996). We refer to thosechanges as “priming” because of the many functional differences thatremain between cold-exposed and thrombin- or ADP-stimulated platelets.Since platelet activation is potentially lethal in coronary and cerebralblood vessels subjected to core body temperatures, we have proposed thatplatelets are thermosensors, designed to be relatively inactive at thecore body temperature of the central circulation but to become primedfor activation at the lower temperatures of external body surfaces,sites most susceptible to bleeding throughout evolutionary history(Hoffmeister et al., 2001). The findings reported here suggest thatirreversible changes in GPIbα are the reason for the clearance of cooledplatelets. Rather than allowing chilled platelets to circulate, theorganism clears low temperature-primed platelets by phagocytosis.

A system involving at least two clearance pathways, one for removal oflocally activated platelets and another for taking out excessivelyprimed platelets (FIG. 7), can possibly explain why chilled plateletscirculate and function normally in CR3-deficient mice and have aslightly prolonged circulation following removal of GPIbα. We proposethat some primed platelets enter microvascular clots on a stochasticbasis. Others are susceptible to repeated exposure to body surfacetemperature, and this repetitive priming eventually renders theseplatelets recognizable by CR3-bearing liver macrophages. Plateletsprimed by chilling are capable of normal hemostatic function inCR3-deficient mice, and coagulation contributes to their clearance.However, the slightly shorter survival time of autologous platelets inCR3-deficient mice examined is probably not ascribable to increasedclearance of normally primed platelets in microvascular clots, becausethe clearance rate of refrigerated platelets was indistinguishable fromthat of platelets kept at room temperature.

References for Background of the Invention and Example 1

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Example 2

Implication of the α_(M)β₂ (CR3) Lectin Domain in Chilled PlateletPhagocytosis.

α_(M)β₂ (CR3) has a cation-independent sugar-binding lectin site,located “C-T” to its I-domain (Thornton et al, J. Immonol. 156,1235-1246, 1996), which binds to mannans, glucans andN-Acetyl-D-glucosamine (GlcNAc). Since CD16b/α_(M)β₂ membrane complexesare disrupted by β-glucan, N-Acetyl-D-galactosamine (GalNAc), andmethyl-α-mannoside, but not by other sugars, it is believed that thisinteraction occurs at the lectin site of the α_(M)β₂ integrin (CR3)(Petty et al, J. Leukoc. Biol. 54, 492-494, 1993; Sehgal et al, J.Immunol. 150, 4571-4580, 1993).

The lectin site of α_(M)β₂ integrin has a broad sugar specifity (Ross,R. Critical Reviews in Immunology 20, 197-222, 2000). Although sugarbinding to lectins is usually of low affinity, clustering can cause amore robust interaction by increasing avidity. The clustering of GPIbαfollowing cooling, as shown by electron microscopy, suggests such amechanism. The most common hexosamines of animal cells are D-glucosamineand D-galactosamine, mostly occuring in structural carbohydrates asGlcNAc and GalNAc, suggesting that the α_(M)β₂ integrin lectin domainmight also bind to mammalian glycoproteins containing carbohydrates thatare not covered by sialic acid. The soluble form of GPIbα, glycocalicin,has a carbohydrate content of 60% comprising N- as well asO-glycosidically linked carbohydrate chains (Tsuji et al, J. Biol. Chem.258, 6335-6339, 1983). Glycocalicin contains 4 potential N-glycosylationsites (Lopez, et al, Proc. Natl. Acad. Sci., USA 84, 5615-5619, 1987).The 45 kDa region contains two sites that are N-glycosylated (Titani etal, Proc Natl Acad Sci 16, 5610-5614, 1987). In normal mammalian cells,four common core structures of O-glycan can be synthesized. All of themmay be elongated, sialylated, fucosylated and sulfated to formfunctional carbohydrate structures. The N-linked carbohydrate chains ofGPIbα are of the complex-type and di-, tri- and tetra-antennarystructures (Tsuji et al, J. Biol. Chem. 258, 6335-6339, 1983). They aresialylated GalNAc type structures with an α(1-6)-linked fucose residueat the Asn-bound GlcNAc unit. There is a structural similarity ofAsn-linked sugar chains with the Ser/Thr-linked: i.e., their position isof a common Gal-GlcNAc sequence. Results suggested that removal ofsialic acid and galactose has no influence on the binding of vWf toglycocalicin, but partial removal of GlcNac resulted in the inhibitionof vWf binding (Korrel et al, FEBS Lett 15, 321-326, 1988). A morerecent study proposed that the carbohydrate patterns are involved inmaintaining an appropriate functional conformation of the receptor,without participating directly in the binding of vWf (Moshfegh et al,Biochem. Biophys. Res. Communic. 249, 903-909, 1998).

A role of sugars in the interaction between chilled platelets andmacrophages has the important consequence that covalent modification,removal or masking of oligosaccharide residues could prevent thisinteraction. We hypothesized that if such prevention does not impairnormal platelet function, we may be able to modify platelets and enablecold platelet storage. Here, we show evidence that favor thishypothesis: 1) Saccharides inhibited phagocytosis of chilled plateletsby macrophages in vitro, and the specific sugars that are effectiveimplicated α-glucans as the relevant targets. Low concentrations ofα-GlcNAc were surprisingly effective inhibitors, consistent with theidea that interference with a relatively small number of clusteredsugars may be sufficient to inhibit phagocytosis. Addition of sugars atconcentrations that maximally inhibited phagocytosis of chilledplatelets has no effect on normal GPIbα function (vWf-binding); 2) Aβ-GlcNAc-specific lectin, but not other lectins, bound avidly to chilledplatelets; 3) Removal of GPIbα or β-GlcNAc residues from plateletsurfaces prevented this binding (since β-GlcNAc removal exposed mannoseresidues, it did not prevent phagocytosis by macrophages which havemannose receptors); 4) Blocking of exposed β-Glucans on chilledplatelets by enzymatic addition of galactose markedly inhibitedphagocytosis of chilled platelets by macrophages in vitro and extendedthe circulation times of chilled platelets in normal animals.

Effect of Monosaccharides on Phagocytosis of Chilled Platelets.

To analyze the effects of monosaccharides on platelet phagocytosis,phagocytes (differentiated monocytic cell line THP-1) were incubated inmonosaccharide solutions at various concentrations, and the chilled orroom temperature platelets were added. Values in the Figures aremeans±SD of 3-5 experiments comparing percentages of orange-positivemonocytes containing ingested platelets incubated with RT or chilledplatelets). While 100 mM D-glucose inhibited chilled plateletphagocytosis by 65.5% (P<0.01), 100 mM D-galactose did not significantlyinhibit chilled platelet phagocytosis (n=3) (FIG. 8A). The D-glucoseα-anomer (α-glucoside) did not have an inhibitory effect on chilledplatelet phagocytosis, although 100 mM inhibited by 90.2% (FIG. 8B) Incontrast, β-glucoside inhibited phagocytosis in a dose-dependent manner(FIG. 8B). Incubation of the phagocytes with 100 mM β-glucosideinhibited phagocytosis by 80% (p<0.05) and 200 mM by 97% (P<0.05),therefore we concluded that the β-anomer is preferred. D-mannose and itsα- and β-anomers (methyl-α-D-mannopyranoside (FIG. 8C) andmethyl-β-D-mannopyranoside (FIG. 8C) had no inhibitory effect on chilledor RT platelet phagocytosis. Incubation of phagocytes using 25 to 200 mMGlcNAc (N-acetyl-D-glucosamine) significantly inhibited chilled plateletphagocytosis. Incubation with 25 mM GlcNac was sufficient to inhibit thephagocytosis of chilled platelets by 86% (P<0.05) (FIG. 8D), whereas 10μM of the β-anomer of GlcNAc inhibited the phagocytosis of chilledplatelets by 80% (p<0.01) (FIG. 8D). None of the monosachharides had aninhibitory effect on RT platelet phagocytosis. Table 2 summarizes theinhibitory effects of monosaccharides at at the indicated concentrationson chilled platelet phagocytosis (**P<0.01, *P<0.05). None of themonosaccharides inhibited thrombin or ristocetin induced human plateletaggregation or induced α-granule secretion as measured by P-selectinexposure.

TABLE 2 Inhibitory effects of monosaccharides on chilled plateletphagocytosis Monosaccharides % inhibition phagocytosis mM D-(+)-glucose65.5  100 D-(+)-galactose — 100 Methyl-α-D- 90.2* 100 glucopyranosideMethyl-β-D- 80.2* 100 gludopyranoside 97.1* 200 D-(+)-mannose — 100Methyl-α-D- — 100 mannopyranoside Methyl-β-D- — 100 mannopyranosideβ-GlcNac 80.9* 0.01 GlcNac 86.3* 25 83.9* 100 83.1* 200Binding of Various Lectins to Room Temperature Platelets or ChilledPlatelets.

β-GlcNAc strongly inhibited chilled human platelet phagocytosis in vitroat μM concentrations, indicating that GlcNac is exposed after incubationof platelets in the cold. We then investigated whether wheat germagglutinin (WGA), a lectin with specificity towards the terminal sugar(GlcNAc), binds more effectively to chilled platelets than to roomtemperature platelets. Washed, chilled or room temperature plateletswere incubated with 2μg/ml of FITC coupled WGA or FITC coupledsuccinyl-WGA for 30 min at room temperature and analyzed by flowcytometry. FIGS. 9A and 9B show the dot plots after incubation withFITC-WGA of room temperature (RT) or chilled (Cold) human platelets. WGAinduces platelet aggregation and release of serotonin or ADP atconcentrations between 25-50 μg/ml WGA (Greenberg and Jamieson, Biochem.Biophys. Acta 345, 231-242, 1974). Incubation with 2 μg/ml WGA inducedno significant aggregation of RT-platelets (FIG. 9A, RT w/WGA), butincubation of chilled platelets with 2 μg/ml WGA induced massiveaggregation (FIG. 9B, Cold/w WGA). FIG. 9C shows the analysis ofFITC-WGA fluorescence binding to chilled or room temperature platelets.To verify that the increase of fluorescence binding is not aggregationrelated, we used succinyl-WGA (S-WGA), a dimeric derivate of the lectinthat does not induce platelet aggregation (Rendu and Lebret, Thromb Res36, 447-456, 1984). FIGS. 9D and 9E show that succinyl-WGA (S-WGA) didnot induce aggregation of room temperature or chilled platelets, butresulted the same increase in WGA binding to chilled platelets versusroom temperature platelets (FIG. 9F). The enhanced binding of S-WGAafter chilling of platelets cannot be reversed by warming of chilledplatelets to 37° C.

Exposed β-GlcNAc residues serve as substrate for aβ1,4glactosyltransferase enzyme that catalyses the linkageGalβ-1GlcNAcβ1-R. In support of this prediction, masking of β-GlcNAcresidues by enzymatic galactosylation inhibited S-WGA binding to coldplatelets, phagocytosis of chilled platelets by THP-1 cells, and therapid clearance of chilled platelets after transfusion into mice. Theenzymatic galactosylation, achieved with bovineβ1,4galactosyltransferase and its donor substrate UDP-Gal, decreasedS-WGA binding to chilled human platelets to levels equivalent to roomtemperature platelets. Conversely, the binding of the galactose-specificRCA I lectin increased by ˜2 fold after galactosylation. UDP-Glucose andUDP alone had no effect on S-WGA or RCA I binding to chilled or roomtemperature human platelets.

We found that the enzymatic galactosylation of human and mouse plateletsis efficient without addition of exogenous β1,4galactosyltransferase.The addition alone of the donor substrate UDP-Gal reduces S-WGA bindingand increases RCA I binding to chilled platelets, inhibits phagocytosisof chilled platelets by THP1 cells in vitro, and prolongs thecirculation of chilled platelets in mice. An explanation for thisunexpected finding is that platelets reportedly slowly releaseendogenous galactosyltransferase activity. A least one form ofβ1,4galactosyltransferases, β4Gal T1, is present in human plasma, onwashed human platelets and in the supernatant fluids of washedplatelets. Galactosyltransferases may associate specifically with theplatelet surface. Alternatively, the activity may be plasma-derived andleak out of the platelet's open canalicular system. In either case,modification of platelet glycans responsible for cold-mediated plateletclearance is possible by simple addition of the sugar-nucleotide donorsubstrate, UDP-Gal.

Importantly, both chilled and non-chilled platelets show the sameincrease in RCA I binding after galactosylation, implying that β-GlcNAcresidues are exposed on the platelet surface independent of temperature.However chilling is a requirement for recognition of β-GlcNAc residuesby S-WGA and by the α_(M)β₂ integrin. We have demonstrated that chillingof platelets induces an irreversible clustering of GPIb. Generallylectin binding is of low affinity and multivalent interactions with highdensity of carbohydrate ligands increases binding avidity. Possibly thelocal densities of exposed β-GlcNAc on the surface of non-chilledplatelets are too low for recognition, but cold-induced clustering ofGPIbα provides the necessary density for binding to S-WGA or the α_(M)β₂integrin lectin domain. We confirmed by S-WGA and RCA-1 binding flowcytometry that UDP-Gal transfers galactose onto murine platelets in thepresence or absence of added galactosyl transferase and documented thatchilled, galactosylated murine platelets circulate and initially survivesignificantly better than untreated room temperature platelets.

Although the earliest recoveries (<2 min) did not differ betweentransfused RT, chilled and chilled, galactosylated platelets,galactosylation abolished an initial platelet loss of about 20%consistently observed with RT platelets.

Galactosylation of murine and human platelets did not impair theirfunctionality in vitro as measured by aggregation and P-selectinexposure induced by collagen related peptide (CRP) or thrombin atconcentrations ranging from maximally effective to three orders ofmagnitude lower. Importantly, the aggregation responses of unmodifiedand galactosylated chilled human platelets to a range of ristocetinconcentrations, a test of the interaction between GPIb and activatedVWF, were indistinguishable or slightly better. The attachment pointsfor N-linked glycans on GPIbα are outside of the binding pocket for VWF.Moreover, mutant GPIbα molecules lacking N-linked glycans bind VFWtightly.

Using FITC labeled lectins with specificities towards β-galactose (R.communislectin/RCA), 2-3 sialic acid (Maackia amurensis lectin/MAA) or2-6 sialic acid (Sambucus Nigra bark lectin/SNA), we could not detectincreased binding after chilling of platelets by flow cytometry (FIG.10), showing that exposure after chilling of platelets is restricted toGlcNAc.

We localized the exposed β-GlcNAc residues mediating α_(M)β₂ lectindomain recognition of GPIbα N-glycans. The extracellular domain of GPIbαcontains 60% of total platelet carbohydrate content in the form of N-and O-glycosidically linked carbohydrate chain. Accordingly, binding ofperoxidase-labeled WGA to GPIbα is easily detectable in displays oftotal platelet proteins resolved by SDS-PAGE, demonstrating that GPIbαcontains the bulk of the β-GlcNAc-residues on platelets, and binding ofWGA to GPIbα is observable in GPIbα immunoprecipitates. UDP-Gal with orwithout added galactosyltransferase diminishes S-WGA binding to GPIbα,whereas RCA I binding to GPIbα increases. These findings indicate thatgalactosylation specifically covers exposed β-GlcNAc residues on GPIbα.Removal of the N-terminal 282 residues of GPIbα from human plateletsurfaces using the snake venom protease mocarhagin, which inhibitedphagocytosis of human platelets by THP-1 cells in vitro, reduces S-WGAbinding to chilled platelets nearly equivalent to S-WGA room temperaturebinding levels. WGA binds predominantly to the N-terminus of GPIbαreleased by mocarhagin into platelet supernatant fluids as a polypeptideband of 45 kDa recognizable by the monoclonal antibody SZ2 specific forthat domain. The glycans of this domain are N-linked. A small portion ofGPIbα remains intact after mocarhagin treatment, possibly because theopen canalicular system of the platelet sequesters it.Peroxidase-conjugated WGA weakly recognizes the residual plateletassociated GPIbα C-terminus after mocarhagin cleavage, identifiable withmonoclonal antibody WM23.

The cold-induced increase in binding of human platelets to α_(M)β₂integrin and to S-WGA occurs rapidly (within minutes). The enhancedbinding of S-WGA to chilled platelets remained stable for up to 12 daysof refrigerated storage in autologous plasma. RCA I binding remainedequivalent to room temperature levels under the same conditions.Galactosylation doubled the binding of RCA I lectin to platelets andreduced S-WGA binding to baseline RT levels. The increase in RCA I anddecrease in S-WGA binding were identical whether galactosylationproceeded or followed storage of the platelets in autologous plasma forup to 12 days. These findings indicate that galactosylation of plateletsto inhibit lectin binding is possible before or after refrigeration andthat the glycan modification is stable during storage for up to 12 days.Platelets stored at room temperature rapidly lose responsiveness toaggregating agents; this loss does not occur with refrigeration.Accordingly, refrigerated platelets with or without galactosylation,before or after storage, retained aggregation responsiveness to thrombinfor up to 12 days of cold storage.

Effects of β-hexosaminidase (β-Hex) and Mocarhagin (MOC) on FITC-WGALectin Binding to Chilled Versus Room Temperature Stored Platelets.

The enzyme β-hexosaminidase catalyzes the hydrolysis of terminalβ-D-N-acetylglucosamine (GlcNAc) and galactosamine (GalNAc) residuesfrom oligosaccharides. To analyze whether removal of GlcNAc residuesreduces the binding of WGA to the platelet surface, chilled and roomtemperature washed human platelets were treated with 100 U/ml β-Hex for30 min at 37° C. FIG. 11A shows the summary of FITC-WGA binding to thesurface of room temperature or chilled platelets obtained by flowcytometry before and after β-hexosaminidase treatment. FITC-WGA bindingto chilled platelets was reduced by 85% after removal of GlcNac (n=3).We also checked whether, as exspected, removal of GPIbα from theplatelet surface leads to reduced WGA-binding after platelet chilling.GPIbα was removed from the platelet surface using the snake venommocarhagin (MOC), as described previously (Ward et al, Biochemistry 28,8326-8336, 1996). FIG. 11B shows that GPIbα removal from the plateletsurface reduced FITC-WGA binding to chilled platelets by 75% and hadlittle influence on WGA-binding to GPIbα-depleted room temperatureplatelets (n=3). These results indicate that WGA binds mostly tooligosaccharides on GPIbα after chilling of human platelets, and it isvery tempting to speculate that the Mac-1 lectin site also recognizesthese exposed sugars on GPIbα leading to phagocytosis.

Masking of Human Platelet GlcNAc Residues by Galactose-transfer GreatlyReduces Their Phagocytosis After Chilling In Vitro and DramaticallyIncreases Their Survival in Mice.

To achieve galactose transfer onto platelets, isolated human plateletswere incubated with 200 μM UDP-galactose and 15 mU/ml galactosetransferase for 30 min at 37° C., followed by chilling or maintenance atroom temperature for 2 h. Galactosylation reduced FITC-WGA bindingalmost to resting room temperature levels. Platelets were fed to themonocytes and platelet phagocytosis was analyzed as described above.FIG. 12 shows that galactose transfer onto platelet oligosaccharidesreduces greatly chilled platelet (Cold) phagocytosis, but does notaffect the phagocytosis of room temperature (RT) platelets (n=3). Theseresults show that in vitro the phagocytosis of chilled platelets can bereduced through coverage of exposed GlcNAc residues. We tested whetherthis approach could be extended to animals and used to increase thecirculation time of chilled platelets. Murine platelets were isolatedand stained with CMFDA. Using the same approach of galactose transferdescribed for human platelets above, wild type murine platelets weregalactosylated and chilled, or not, for 2 hours. 10⁸ Platelets weretransfused into wild type mice and their survival determined. FIG. 13shows the survival of these chilled, galactosylated murine plateletsrelative to untreated platelets. Both platelets kept at room temperature(RT) and the galactosylated chilled platelets (Cold+GalT) had almostidentical survival times, whereas chilled untreated platelets (Cold)were cleared rapidly as expected. We believe galactosylated chilledplatelets will circulate in humans.

We noted that our control reaction, in which galactose transferase washeat-inactivated also resulted in glycan modification of platelets asoccurred in the experimental reaction with active galactose transferase,as judged by WGA binding (FIG. 14A), in vitro phagocytosis results inhuman platelets (FIG. 14B), and survival of murine platelets (FIG. 14C).Therefore, we conclude that platelets contain galactose transferaseactivity on their surface, which is capable of directing glycanmodification using only UDP-galactose without the addition of anyexogenous galactose transferase. Thus, glycan modification of plateletscan be achieved simply by incubation of UDP-galactose.

UDP-galactose Incorporate into Human Platelets in a Time DependentMatter.

In another set of experiments we have shown that ¹⁴C-labeledUDP-galactose incorporates into human platelets in a time dependentmanner in the presence or absence of the enzyme galactosyl transferase.FIG. 15 shows the time course of ¹⁴C-labeled UDP-galactose incorporationinto washed human platelets. Human platelets were incubated with¹⁴C-labeled UDP-galactose for different time intervals in the absence ofgalactosyl transferase. The platelets were then washed and the ¹⁴Cradioactivity associated with platelets measured.

Example 3

Enzymatic Modification of Platelet β-glycans Inhibit Phagocytosis ofCooled Platelets by Macrophages In Vitro and Accommodate NormalCirculation In Vivo.

Our preliminary experiments have demonstrated the enzymatic covering ofGlcNAc residues on GPIbα using galactose-transfer (glycan modification)onto chilled human platelet surfaces greatly reduced their in vitrophagocytosis. One interpretation of these findings is that GPIbαstructure is altered on the surface of chilled human and murineplatelets. This causes the exposure or clustering of GlcNAc, which isrecognized by the lectin binding domain of αMβ2 leading to plateletremoval. β-GlcNAc exposure can be measured by WGA binding and possiblyby binding of recombinant αMβ2 lectin domain peptides. Resting humanplatelets bind WGA, which increases greatly after chilling. We proposethat galactose transfer (glycan modification) will prevent GPIbα'sinteraction with αMβ2-lectin but not with vWf. This modification(galactose transfer onto platelet surface) leads to normal survival ofchilled platelets in WT mice as shown by our preliminary experiments.

Example 4

This example shows that the αMβ2 lectin site mimics WGA and sugarmodifications prevent the engagement of the recombinant lectin site withchilled platelets. Dr. T. Springer (Corbi, et al., J. Biol Chem. 263,12403-12411, 1988) provided the human αM cDNA and several anti-αMantibodies. The smallest r-huαM construct exhibiting lectin activitythat has been reported includes its C-T and a portion of its divalentcation binding region (residues 400-1098) (Xia et al, J Immunol 162,7285-7293, 1999). The construct is 6×His-tagged for ease ofpurification. We first determined if the recombinant lectin domain canbe used as a competitive inhibitor of chilled platelet ingestion in thephagocytic assay. Competition proved that the αM lectin site mediatesbinding to the platelet surface and initiates phagocytosis. As controls,a construct lacking the lectin-binding region of αM was used and therecombinant protein was denatured. Lectin binding domain functions as aspecific inhibitor of chilled platelet ingestion. We made a αM constructthat include GFP and express and labeled the αM-lectin binding site withFITC and used it to label the surface of chilled platelets by flowcytometry. Platelets were labeled with CMFDA. We found that chilledplatelets bind more efficiently to the αM lectin side of αMβ2 integrincompared to room temperature paltelets. The lectin side and wholeαM-construct (Mac-1) was expressed in Sf9 insect cells.

The platelet sugar chains are modified to inhibit theplatelet-oligosaccharide interaction with the r-huαM-lectin site. Theefficiency of sugar modifications is also monitored by inhibition of thebinding of fluorescent-lectin domain binding to platelets by flowcytometry.

The recovery and circulation times of room temperature, chilled andchilled-modified platelets are compared to establish that galactosetransfer onto chilled murine platelets results in longer circulatingplatelets. Room temperature, chilled and chilled-modified platelets arestained with CMFDA, and 10⁸ platelets transfused into wild type mice asdescribed above. The mice are bled immediately (<2 min.), 30 min, 1 h,2, 24, 48 and 72 hours after transfusion. The blood obtained is analyzedusing flow cytometry. The percentage of fluorescent labeled plateletswithin the gated platelet population measured immediately afterinjection is set as 100%. The recovery of fluorescently labeledplatelets obtained at the various time points is calculated accordingly.

Example 5

This example demonstrates that chilled, unmodified and chilled,galactosylated (modified) platelets have hemostatic function in vitroand in vivo. Chilled platelets are not “activated” in the sense ofagonist-stimulated platelets. Patients undergoing surgery underhypo-thermic conditions may develop thrombocytopenia or show severehemostatic post-operative impairments. It is believed that under thesehypothermic conditions, platelets might lose their functionality.However, when patients undergo hypothermic surgery, the whole organismis exposed to hypothermia leading therefore to changes in multipletissues. Adhesion of non-chilled platelets to hepatic sinusoidalendothelial cells is a major mechanism of cold preservation injury(Takeda, et al. Transplantation 27, 820-828, 1999). Therefore, it islikely that it is the interaction between cold hepatic endothelium andplatelets, not platelet chilling per se, that leads to deleteriousconsequences under hypothermic conditions of surgery or trans-plantationof cold preserved organs (Upadhya et al, Transplantation 73, 1764-1770,2002). Two approaches showed that chilled platelets have hemostaticfunction. In one approach, the circulation of chilled platelets inαMβ2-deficient mice facilitates studies of platelet function aftercooling. In the other approach, the function of modified chilled and(presumably) circulating platelets was tested.

Human and murine unmodified and modified (galactosylated) chilledplatelets were tested for functionality, including in vitro aggregationto agonists, P-selectin exposure and fibrinogen binding.

αMβ2 deficient or WT mice are transfused with murine chilled/RTplatelets modified or not, and allowed to circulate for 30 min., 2 and24 hours. We determine if chilled platelets contribute to clottingreactions caused by tail vein bleeding and if these platelets bindagents such as fibrinogen after activation. We also determine howchilled platelets, modified or not, contribute to clotting on ferricchloride injured and exteriorized mouse mesenteries, an in vivothrombus-formation model that we developed. This method detects thenumber of platelets adherent to injured vessels and has documentedimpaired platelet vessel wall interactions of platelets lackingglycoprotein V or β3-integrin function (Ni et al, Blood 98, 368-3732001; Andre, et al. Nat Med 8, 247-252, 2002). Last, we determine thestorage parameters of the modified platelets.

In vitro platelet function is compared using aggregation with thrombinand ADP and botrocetin induced vWf-binding to murine platelets. Murineand human chilled platelets modified (galactosylated) or unmodifiedplatelets are normalized to a platelet concentration of 0.3×10⁹/mm³, andaggregation induced using the various agonists according to standardprotocols (Bergmeier, et al. 2001 276, 25121-25126, 2001). To studyvWf-binding we activate murine vWf using botrocetin and analyze thebinding of fluorescently labeled vWf to chilled platelets modified ornot in PRP (Bergmeier, et al. 2001 276, 25121-25126, 2001). To evaluatewhether degranulation of platelets occurs during modification, we alsomeasure P-selectin exposure of chilled murine and human plateletsmodified or not using fluorescent labeled anti-P-selectin antibodies byflow cytometry (Michelson et al., Proc. Natl. Acad. Sci., USA 93,11877-11882, 1996).

10⁹ CMFDA-labeled platelets are transfused into mice, first verifyingthat these platelets are functional in vitro. We determine whetherchilled platelets contribute to aggregation by transfusing chilled orroom temperature CMFDA-labeled platelets into αMβ2 deficient mice. At 30min., 2 hours and twenty-four hours after the infusion of platelets, astandard tail vein bleeding test is performed (Denis, et al. Proc NatlAcad Sci USA 95, 9524-9529, 1998). The emerging blood is fixedimmediately in 1% formaldehyde and platelet aggregation is determined bywhole blood flow cytometry. Platelet aggregates appear as bigger sizedparticles in the dot plot analysis. To verify that the transfusedplatelets do not aggregate in the normal circulation we also bleed themice through the retroorbital eye plexus into an anticoagulant.Platelets do not form aggregates under these bleeding conditions. Theemerging blood is fixed immediately and platelets are analyzed by flowcytometry in whole blood as described above. Platelets are identifiedthrough binding of a phycoerythrin-conjugated α_(IIb)β₃ specificmonoclonal antibody. The infused platelets in the blood sample areidentified by their CMFDA-fluorescence. Non-infused platelets areidentified by their lack of CMFDA fluorescence (Michelson, et al, Proc.Natl. Acad. Sci., U.S.A. 93, 11877-11882, 1996). The same set of testsis performed with CMFDA modified (galactosylated) chilled plateletstransfusing these platelets into αMβ2 and WT. This experiment testsaggregation of chilled platelets modified or not in shed blood.

10⁹ CM-orange labeled unmodified chilled or room temperature plateletsare transfused into αMβ2 deficient mice to verify that these plateletsare functional in vitro. At 30 min., 2 h and twenty-four hours after theinfusion of CM-orange labeled platelets, PRP is isolated as describedand analyzed by flow cytometry. P-selectin exposure is measured using ananti FITC-conjugated anti P-selectin antibody (Berger, et al, Blood 92,4446-4452, 1998). Non-infused platelets are identified by their lack ofCM-orange fluorescence. The infused platelets in the blood sample areidentified by their CM-orange fluorescence. CM-orange and P-selectinpositive platelets appear as double positive fluorescently(CM-orange/FITC) stained platelets. To verify that chilled plateletsstill expose P-selectin after thrombin activation, PRP is activatedthrough the addition of thrombin (1 U/ml, 2 min at 37° C.) andP-selectin exposure is measured as described. To analyze the binding offibrinogen to α_(IIb)β₃, isolated platelets are activated through theaddition of thrombin (1 U/ml, 2 min, 37° C.) and Oregon-green coupledfibrinogen (20 μg/ml) added for 20 min at 37° C. (Heilmann, et al,Cytometry 17, 287-293, 1994). The samples are analyzed immediately byflow cytometry. The infused platelets in the PRP sample are identifiedby their CM-orange fluorescence. CM-orange and Oregon-green positiveplatelets appear as double positive fluorescently stained(CM-orange/Oregon green) platelets. The same sets of experiments areperformed with CM-orange labeled modified (galactosylated) chilledplatelets transfused into αMβ2 deficient and WT mice.

Example 6

In Vivo Thrombosis Model

First, we show the delivery of RT and unmodified chilled platelets toinjured endothelium of αMβ2 deficient mice using double fluorescentlylabeled platelets. The resting blood vessel is monitored for 4 min.,then ferric chloride (30 μl of a 250-mM solution) (Sigma, St Louis, Mo.)is applied on top of the arteriole by superfusion, and video recordingresumed for another 10 min. Centerline erythrocyte velocity (Vrbc) ismeasured before filming and 10 min after ferric chloride injury. Theshear rate is calculated on the basis of Poiseuille's law for aNewtonian fluid (Denis, et al, Proc Natl Acad Sci USA 95, 9524-9529,1998). These experiments show if chilled platelets have normalhemostatic function. We repeat these experiments in WT mice comparing RTand galactosylated chilled platelets using two different, fluorescentlylabeled platelet populations injected into the same mouse and analyzethe thrombus formation and incorporation of both platelet populations.

We then compare in vitro platelet functions and survival and in vivohemostatic activity of chilled and modified chilled murine plateletsstored for 1, 5, 7 and 14 days under refrigeration as described above.We compare the recovery and circulation times of these stored chilledand modified chilled platelets and prove that: 1) the modificationthrough galactose transfer onto chilled murine platelets is stable afterthe long term refrigeration; and 2) that these platelets functionnormally. Survival experiments are performed as described above. We useWGA binding, to verify that GlcNAc residues remain covered by galactoseafter the longer storage time points. As an ultimate test that thesemodified, stored platelets are functionally intact and contribute tohemostasis, we transfuse them into total-body-irradiated mice (Hoyer, etal, Oncology 49, 166-172, 1992). To obtain the sufficient numbers ofplatelets, we inject mice with commercially available murinethrombopoietin for seven days to increase their platelet count (Lok, etal. Nature 369, 565-558, 1994). Isolated platelets are modified usingthe optimized galactose transfer protocol, stored under refrigeration,transfused, and tail vein bleeding times measured. Since unmodifiedchilled platelets do not persist in the circulation, a comparison ofmodified cooled platelets with room temperature stored platelets is notnecessary at this point. The murine platelets are stored underrefrigeration in standard test tubes. If a comparison with roomtemperature stored murine platelets is necessary we switch to primateplatelets. Rather than engineer special down-scale, gas-permeablestorage containers to accommodate mouse platelets, such comparisons aremore appropriate for primates (including humans) for which roomtemperature storage bags have been designed.

Example 7

Galactosylation of Platelets in a Platelet Concentrate.

Four different platelet concentrates were treated with increasingconcentrations of UDP galactose: 400 μM, 600 μM, and 800 μM. RCA bindingratio measurements showed a dose dependent increase in galactosylationin the four samples tested. (FIG. 16). Our results provide evidence thatgalactosylation is possible in platelet concentrates.

It should be understood that the preceding is merely a detaileddescription of certain preferred embodiments. It therefore should beapparent to those skilled in the art that various modifications andequivalents can be made without departing from the spirit and scope ofthe invention. It is intended to encompass all such modifications withinthe scope of the appended claims.

All references, patents and patent publications that are recited in thisapplication are incorporated in their entirety herein by reference.

1. A device for collecting and processing platelets comprising: acontainer for collecting platelets; at least one satellite container influid communication with the container; and at least one glycanmodifying agent in the at least one satellite container, wherein the atleast one glycan modifying agent is UDP-galactose.
 2. The device ofclaim 1, wherein the at least one glycan modifying agent in thesatellite container is present in sufficient amounts to preserve aplurality of platelets collected in the container for collectingplatelets.
 3. The device of claim 2, wherein the satellite container isconnected to the container by a breakable seal.
 4. The device of claim1, wherein the container for collecting platelets further comprisesplasma containing a plurality of platelets.
 5. The device of claim 1further comprising at least one additional glycan modifying agent in theat least one satellite container, wherein the at least one additionalglycan modifying agent is selected from the group consisting of:D-glucose, methyl-α-D-glucopyranoside, methyl-β-D-glucopyranoside,N-acetyl-β-glucosamine, and N-acetyl-glucosamine.